AU711851B2

AU711851B2 - Identification of genes altered in multiple myeloma

Info

Publication number: AU711851B2
Application number: AU32173/97A
Authority: AU
Inventors: Riccardo Dalla-Favera
Original assignee: Columbia University of New York
Current assignee: Columbia University of New York
Priority date: 1996-05-28
Filing date: 1997-05-28
Publication date: 1999-10-21
Anticipated expiration: 2017-05-28
Also published as: EP1731144A1; US6958386B1; US6245562B1; AU3217397A; DE69736476D1; JP2000512133A; WO1997045106A1; EP0920305A1; DK0920305T3; US20070141579A1; DE69736476T2; CA2256455A1; ATE335470T1; EP0920305A4; EP0920305B1

Abstract

This invention provides a method of determining a chromosomal breakpoint in a subject suffering from multiple myeloma which comprises steps of: (a) obtaining a DNA sample from the subject suffering from multiple myeloma; (b) determining whether there is J and C disjunction in the immunoglobulin heavy chain gene in the obtained DNA sample; (c) obtaining a genomic library having clones which contain genomic DNA fragments from the DNA sample which shows positive J and C disjunction; (d) selecting and isolating clones of the obtained library which show positive hybridization with a probe which is capable of specifically hybridizing with the C but not the J region of the immunoglobulin heavy chain gene; (e) preparing fluorescent probes from the genomic DNA fragments of the isolated clones from step (d); (f) hybridizing said fluorescent probes with metaphase chromosomes; and (g) determining the identity of the chromosomes which are capable of hybridizing to said fluorescent probes, wherein the identification of a chromosome other than chromosome 14 would indicate that the chromosomal breakpoint is between chromosome 14 and the identified chromosome, thereby determining a chromosomal breakpoint in a subject suffering from multiple myeloma. This invention also provides the identified gene altered by a chromosomal breakpoint and various uses thereof.

Description

WO 97/45106 PCT/US97/09065 IDENTIFICATION OF GENES ALTERED IN MULTIPLE MYELOMA The invention disclosed herein was made with Government support under NIH Grant No. CA 44025, CA-44029, and CA- 34775. Accordingly, the U.S. Government has certain rights in this invention.

Background of the Invention Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.

Multiple myeloma (MM) is an incurable B cell tumor affecting B cell end-stage differentiation. Clinically, the course of MM is similar to end-stage plasma cell leukemia (PCL), i.e., there is an uncontrollable proliferation of myeloma cells accompanied by numerous complications, including hyperviscosity syndromes, hypercalcemia, infections, multiple bone fractures, and organ failure.

Non-random chromosomal translocation is known to play a crucial role in the tumorigenesis of hematologic malignancies In B-cell lymphomas, many important proto-oncogenes deregulated by juxtaposition to immunoglobulin (Ig) gene locus have been identified. Each proto-oncogene is associated with a specific subtype of lymphoma, such as c-MYC in Burkitt's lymphoma, Cyclin DI IBCLI in mantle cell lymphoma, BCL-2 in follicular lymphoma and BCL-6 in diffuse large cell lymphoma In WO 97/45106 PCT/US97/09065 -2contrast, little is known about molecular alterations of human MM/PCL, due to the difficulty in cytogenetic analysis.

However, previous cytogenetic reports have shown a 14q+ chromosome, suggesting the existence of a chromosomal translocation involving the Ig heavy chain (IgH) locus, is observed in 20 30 of the MM/PCL cases and it is the most frequent consistent abnormality Even in such cases, most cytogenetic data have failed to identify donor chromosomes other than llq13, 8q24, and 18q21, where protooncogenes Cyclin DIIBCL-IIPRADI, c-MYC and BCL-2 are located, respectively. Among them, the 11q13 locus has been demonstrated to be involved in nearly 5~10% of the cases and also in 62% of the established cell lines (13) The t(ll;14) (q13;q32) translocation is also accompanied by a corresponding overexpression of the Cyclin D1 gene, which raises a strong possibility of the involvement of this gene, although the breakpoints at llq13 do not cluster like those of the lymphoma cases (14-16). Recent advances in fluorescence in situ hybridization (FISH) have made it possible to clarify both the frequency of the 14q+ chromosomes and the partner chromosomes of the IgH loci.

One such report revealed an intriguing result, that numerous chromosomal loci are able to translocate to IgH locus, including 6p21, lq21, 3pll, 7qll, 11q23 This has prompted a search for the proto-oncogenes deregulated by the regulatory elements of the IgH gene for a further understanding of the molecular mechanisms of MM/PCL. In the present study, one candidate proto-oncogene, MUM1 (multiple myeloma oncogene was found juxtaposed to the IgH gene as a result of t(6;14)(p25; q32) translocation in human myeloma cell line, SKMM-1. Over expression of the MUM1 mRNA was observed in this cell line. A second gene, called MUM-2 I WO 97/45106 PCT/US97/09065 -3was found translocated in proximity to the IgH gene on chromosome 14q32 in human myeloma cell line, U-266.

The method of analysis of 14q+ chromosomal translocations and identification of the genes altered in multiple myeloma of this invention are useful since 1) no method is currently available to determine the chromosomal sequences involved in 14q+ translocations, the most important cytogenetic lesions associated with MM pathogenesis; 2) no specific gene lesion is currently known for MM; 3) no diagnostic method based on gene/DNA lesion is currently available for MM and 4) there are no therapeutic approaches aimed at counteracting the action of abnormal gene products in MM.

WO 97/45106 PCT/US97/09065 -4- Summary of the Invention This invention provides a method of determining a chromosomal breakpoint in a subject suffering from multiple myeloma which comprises steps of: obtaining a DNA sample from the subject suffering from multiple myeloma; (b) determining whether there is J and C disjunction in the immunoglobulin heavy chain gene in the obtained DNA sample; obtaining a genomic library having clones which contain genomic DNA fragments from the DNA sample which shows positive J and C disjunction; selecting and isolating clones of the obtained library which show positive hybridization with a probe which is capable of specifically hybridizing with the C but not the J region of the immunoglobulin heavy chain gene; preparing fluorescent probes from the genomic DNA fragments of the isolated clones from step hybridizing said fluorescent probes with metaphase chromosomes; and determining the identity of the chromosomes which are capable of hybridizing to said fluorescent probes, wherein the identification of a chromosome other than chromosome 14 would indicate that the chromosomal breakpoint is between chromosome 14 and the identified chromosome, thereby determining a chromosomal breakpoint in a subject suffering from multiple myeloma.

This invention provides a method to identify a gene other than the immunoglobulin gene which is located in chromosome 14, altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma which comprises steps of: a) selecting a probe having a sequence of a chromosome other than chromosome 14, identified at the chromosomal breakpoint detected in a subject suffering from multiple myeloma, wherein said probe is capable of hybridizing to the unique sequence of the gene other than I WO 97/45106 PCTIUS97/09065 the immunoglobulin gene altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma; b) contacting said probe with mRNA isolated from a cell under conditions permitting formation of a complex between said probe and the mRNA; c) isolating the complex resulting from step d) determining the sequence of the mRNA in the isolated complex, thereby determining the identity of the gene.

This invention provides a gene designated MUM-I. This invention provides a gene designated MUM-2. This invention provides an isolated nucleic acid molecule encoding a MUM protein. This invention provides a DNA encoding a MUM protein. This invention provides a cDNA encoding a MUM protein. This invention provides a genomic DNA molecule encoding a MUM protein. This invention provides a RNA molecule encoding a MUM protein. This invention provides an isolated nucleic acid molecule encoding a human MUM-l protein. This invention provides an isolated nucleic acid molecule encoding a human MUM-2 protein. This invention provides an isolated nucleic acid molecule encoding a MUM protein operatively linked to a promoter of RNA transcription. This invention provides a vector comprising the an isolated cDNA encoding a MUM protein. This invention provides a vector which comprises an isolated cDNA encoding a MUM protein. This invention provides a vector which comprises an isolated cDNA encoding a MUM protein, wherein the vector is a plasmid. This invention provides a host cell for the vector which comprises an isolated cDNA encoding a MUM protein.

This invention provides a nucleic acid probe comprising a WO 97/45106 PCT/US97/09065 -6nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a MUM protein. This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM protein.

This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

This invention provides a nucleic acid probe comprising a the sequence of a nucleic acid molecule encoding a MUM-I protein which is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14.

This invention provides a nucleic acid probe comprising a the sequence of a nucleic acid molecule encoding a MUM-2 protein which is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14.

This invention provides a method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-i protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-i protein. This invention provides a method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-2 protein in a sample from a subject which comprises detecting in a sample from the -M -N 1- WO 97/45106 PCTIUS97/09065 -7subject a rearrangement of nucleic acid encoding MUM-2 protein.

This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-1 protein so as to prevent overexpression of the mRNA molecule. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-2 protein so as to prevent overexpression of the mRNA molecule.

This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an isolated cDNA molecule encoding a MUM protein. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the isolated genomic DNA molecule encoding a MUM protein. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an isolated RNA molecule encoding a MUM protein.

This invention provides a purified MUM protein. This invention provides a purified MUM-1 protein. This invention provides an antibody directed to a purified MUM-1 protein.

This invention provides an antibody capable of specifically recognizing MUM-1 protein. This invention provides a purified MUM-2 protein. This invention provides an antibody directed to a purified MUM-2 protein. This invention provides an antibody capable of specifically recognizing a MUM-2 protein.

This invention provides a pharmaceutical composition WO 97/45106 PCT/US97/09065 -8comprising an amount of an oligonucleotide effective to prevent overexpression of a human MUM-1 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane. This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide effective to prevent overexpression of a human MUM-2 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

WO 97/45106 PCTIUS97/09065 -9- Brief Description of the Figures Figure 1. JH-Cp dissociation in BamHI digested DNA of the 14q+ SK-MM-1 cell line. A 10 p g of the high molecular weight DNA was completely digested with BamHI, loaded on each lane and blotted. The same filter was sequentially hybridized with JH, Cp, Cy 2 and 0.7B/H probes. JH probe detects two rearranged bands of 12.0 kb and 9.7kb. The 9.7 kb band is comigrated with that probed with Cy 2 probe, suggesting it to be a physiological rearrangement. On the other hand, one allele of the Cp locus is deleted and another is rearranged (6.5 kb) without being comigrated with rearranged bands of JH.

Therefore, 12.0 kb and 6.5 kb bands detected by JH and Cp (shown by arrowheads) might represent unknown derivative chromosome and derivative 14 chromosome, respectively. As expected, 0.7B/H probe (Fig. 2A) detected the rearranged band comigrated with band of Cp. Dashed lines show the comigration. Size markers of A/HindIII are shown on the left.

Molecular cloning of the breakpoints of the t(6;14) translocation and germline walking at MUM1 locus. Restriction maps of XSKB-4a and XSKS-3 clones representing derivative 6 and 14 are shown, together with germline maps of IgH locus at 14q32 and MUM1 Figures 2A-B.

WO 97/45106 PCT/US97/09065 Figure 3.

locus at 6 p 25 Arrows indicate the chromosomal breakpoints. B, BamHI; E, EcoRI; H, HindIII. Comparison of the nucleotide sequences around the breakpoints on derivative 6 (SEQ ID NO: 16) and derivative 14 (SEQ ID NO: 17) chromosome. Homologous regions are indicated by dashes. The arrow indicates the breakpoint (SEQ ID NO: Nucleotide (SEQ ID NO: 17) numbers shown below are the same as in the Sp sequence reported by Sun, et al. (18).

Mapping of the MUM1 locus to chromosome 6p25. XMUM-3 genomic clone (Figure 2A) was used as a probe for in situ hybridization.

The white arrow indicates the fluorescence signal on chromosome 6 band p25. Right panel shows the G-banding picture stained with

DAPI.

Expression of the MUM1 gene in hematopoietic lineage. A 10 ug aliquot of total RNA was loaded on each lane and Northern blot analysis was performed using the 2.1H probe (Figure 2A). GAPDH or 3-actin probes were used to control for amount of RNA loaded.

MUM1 RNA expression in various hematopoietic cell lines. MUM1 RNA is detected in B cell and mature T cell lines as a single 6kb transcript. HELA, epithelial lineage; LCL, Epstein-Barr virus-transformed lymphoblastoid cell line; RAMOS and SK-MM-1, Figures 4A-C.

WO 97/45106 PCT/US97/09065 -11- B-cell lineage; HUT-78 and MOLT-4, T-cell lineage; HL-60 and U937, myelomonocytic lineage; K562, erythroid lineage. Dashes indicate 28S and 18S. Expression in B cell lines derived from various stages of B cell differentiation. MUM1 RNA is seen throughout the B cell development except for BJAB cell line. 697, pre-B cell stage; RAMOS and BJA-B, Burkitt cell line representing mature-B cell stage; RPMI-8226 and U-266, plasma cell stage. Comparison of the expression level among myeloma cell lines.

MUM1 RNA is overexpressed in SK-MM-1 cell line carrying t(6;14). Overexpression of the MUM1 is also demonstrated in XG-4, XG-7, and XG-10 cell lines. RPMI-8226, U-266, EJM, and SKMM-1 are IL-6 (interleukin-6) independent lines, whereas XG-1, XG-2, XG-4, XG-6, XG-7, and XG-10 are IL-6 dependent lines.

Figures 5A-B-3. Sequence of MUM1 cDNA and structure of its predicted protein product. (A) Restriction map of the MUM1 cDNA and the position of the open reading frame (box). The solid box indicates approximate position of the DNA binding domain. Sc, SacII; A, ApaI; P, PstI; H, HindIII; S, SacI Nucleotide sequence of the MUM1 cDNA (SEQ ID NO: 13) and corresponding amino acid sequence (SEQ ID NO: 14). Putative WO 97/45106 PCT/US97/09065 Figures 6A-B.

-12translation initiation codons and preceding stop codons appearing in frame are underlined. The asterisk indicates the translation stop codon.

Homology between MUM1 (SEQ ID NO: 1) and other IRF family proteins (SEQ ID NOS: 2-7).

Similarity at N-terminal DNA binding domain. Black background indicates identical residues found more than four times. Gray indicates conserved residues that appear in at least four sequences at a given position. Conserved tryptophan residues in DNA binding domain among IRF family members are indicated by closed circles. Similarity at C-terminal region between human MUM1 (SEQ ID NO: 8), Mouse LSIRF/Pip (SEQ ID NO: Human ICSBP (SEQ ID NO: 10), Human ISGF3y (SEQ ID NO: 11), and Human IRF-3 (SEQ ID NO: 12). Black and gray background are as in Genomic organization of the MUM1 gene and location of the chromosomal breakpoints in multiple myeloma. Filled boxes indicate the coding regions and empty boxes indicate the noncoding regions. The position, and the size of each exon of the MUM1 gene are approximate and have been determined by the hybridizations. One exon in each restriction fragment may consist of more than two exons. Translation initiation Figure 7.

WO 97/45106 PCT/US97/09065 Figure 8.

Figure 9A-B.

-13codon (ATG) and stop codon (TGA) are indicated. Genomic probes used for further investigations are shown as solid bars below the map. Arrows indicate the chromosomal breakpoints of SKMM-1 cell line and case B, BamHI; E, EcoRI; H, HindIII.

Scheme of the t(6;14)(p25;q32) translocation involving the MUM1 and the immunoglobulin heavy chain (IgH) gene loci. VH-D-J-CH indicates variable-diversity joiningconstant region of the IgH gene. Direction of the MUM1 gene on the chromosome 6 is tentatively drawn.

Demonstration of JH-Ca disjunction in U-266 cells and cloning of normal and 14q+ chromosomal breakpoints. The panel shows the results of Southern blot analysis of BamHI digested U-266 and normal control (placenta) genomic DNA using the indicated JH and Ca probes. The arrowheads indicate two DNA fragments containing Ca sequences not linked to JH sequences, suggesting the presence of a chromosomal breakpoint in 14q32. The panel provides a schematic representation of the phage clones isolated from a library constructed from U-266 DNA and screened with a Ca probe. Based on restriction enzyme analysis, the three cloned regions represent a normal Ca region (14q32 germ-line), and two rearranged WO 97/45106 PCTIUS97/09065 -14- Figure 10.

regions (der.14 and 14q32) containing unknown sequences linked to Ca sequences.

The 2.5BE probe used for Northern blot analysis of MUM2 transcripts (Fig. 10) is also shown.

Identification of MUM2 RNA transcripts. The figure shows the results of a Northern blot analysis of RNA extracted from various MM/PCL cell lines using the 2.5BE probe (see Fig. 9) or GAPDH probe (as a control for RNA loading). A 1.9 Kb RNA transcript is detectable in some cell lines including

U-

266, indicating that the 2.5BE fragments represents part of a gene, MUM2.

Schematic representation of IgH DNA rearrangements in normal B cells and in tumors carrying chromosomal translocations breaking the S region of the IgH locus.

Note that in physiological IgH rearrangements (panel 11A) JH sequences and C sequences (Cp before and Cy after switch recombination, respectively) are consistently found within the same BamHI restriction fragment. Conversely, JH and C sequences are not linked, and are present on two different chromosomes [derivative X and derivative 14(14q+)] in cells carrying a chromosomal translocation breaking the switch region (panel 11B) Figure 11A-B.

WO 97/45106 PCT/US97/09065 Figure 12A-B.

Figure 13.

Fig. 14A-B.

MUM1 cDNA. cDNA insert is cloned into EcoRI/BamHI site of the pBluescript KS+.

Bacteria strain used is DH5a cells.

pcMUM1.16a contains full length open reading frame of nt. 217-1572.

Breakpoint Cloning of the U-266 Cell Line.

pMUM2-8 has a 22.0 KB insert in BamHI site of pBluescript KS+.

Molecular cloning of the 14q+ chromosome in SK-MM-1 cell line. A. Southern blot analysis indicating illegitimate rearrangement in IgH locus. A 10 pg of high molecular weight DNA was completely digested with BamHI, loaded on each lane and blotted. The same filter was sequentially hybridized with JH, Cp Cy2, and 0.7B/H (Fig. 14B) probes.

Illegitimately rearranged bands detected by JH and Cp probes are shown by arrowheads.

0.7B/H probe detected the rearranged band comigrated with 6.5kb band detected with Cp probe.-Dashed lines show the comigration.

Size markers of X/HindIII are shown on the left. B. SK-MM-1 cells carry cryptic t(6;14)(p25;q32). Restriction maps of XSKB-4a and XSKS-3 clones representing der and der (14) are shown, together with germline maps of IgH locus at 14q32 and MUM1 locus at 6p25. Genomic organization of the MUM1 gene is shown. Filled boxes indicate the coding regions and empty boxes indicate WO 97/45106 PCT/US97/09065 Fig. 15A-D.

-16the noncoding regions. The position, and the size of each exon of the MUM1 are approximate and have been determined by the hybridizations. One exon in each restriction fragment may consist of more than two exons.

Translation initiation codon (ATG) and stop codon (TGA) are indicated. Genomic probes applied for Southern analyses are shown as solid bars below the map. Arrows indicate the chromosomal breakpoint of the SK-MM-1 cell line. B, BamHI; E, EcoRI; H, HindIII.

A-B. Mapping MUM1 locus to chromosome 6 p 2 in a metaphase spread of normal lymphocytes by 360kb non-chimeric YAC, y927E3 DNA. The arrowheads indicate the fluorescence signals on chromosome 6 band p25. C-D. Splitting of y927E3 DNA signal to chromosome 6p25 and 14q32 in a metaphase spread of XG-7 cell line. The arrowheads indicate positive signals. Right panel shows the corresponding DAPI image showing a G-banding like staining.

Expression of MUM1. A 10 pg aliquot of total RNA was loaded on each lane and Northern blot analysis was performed using 2.1H probe (Fig.

14B). GAPDH or -ACTIN probes were used to control for amount of RNA loaded. A. Left panel shows the distribution in various hematopoietic cell lines. B. Right panel shows expression in B-cell lines derived from various stages of differentiation. HELA, Fig. 16A-E WO 97/45106 PCT/US97/09065 -17epithelial lineage; LCL, Epstein-Barr virus-transformed lymphoblastoid cell line; HUT-78 and MOLT-4, T-cell lineage; HL-60 and U937, myelomonocytic lineage; K562, erythroid lineage; 697, pre-B cell stage; RAMOS and BJA-B, Burkitt lymphoma derived cell lines representing mature-B cell stage. Dashes indicate 28S and 18S. C-D. Comparison of the expression level among MM cell lines. D.

indicates a densitometric quantitation of the MUM1 mRNA corrected by 3-ACTIN level. Mean values of black dots were indicated by horizontal bars. The expression level in SK-MM-1 and XG-7 cell line was indicated by open dot. IL-6 independent MM cell lines, IL-6 dependent MM cell lines.

E. Identification of DNA-binding complexes containing MUM1 in nuclear extracts from MM cell lines. 1 pg of the nuclear extract from MM cell lines was assayed for binding to GBP-ISRE probe by EMSA. Arrows indicate MUM1-containing supershifted complexes performed by using an anti-MUMl/ICSAT antiserum. An anti-BCL6 antiserum was used as a control for specificity. Cold cognate oligomer competition was performed using a 100-fold excess of the GBP-ISRE probe.

Fig. 17A-B. A. Western blot analysis of Rat-1 cell clones with anti-HA (12CA5) MoAb. The 52 kD MUM1-HA product is shown by an arrowhead. B.

Anchorage independent growth of Rat-i cells expressing MUM1 protein. The number of the colonies in soft agar assay of Rat-i cell clones expressing MUM1-HA and those WO 97/45 106 PCT/US97/09065 -18transfected with CMV control vector are shown.

Error bar indicates +1SD.

WO 97/45106 PCT/US97/09065 -19- Detailed Description of the Invention The following standard abbreviations are used throughout the specification to indicate specific nucleotides: C=cytosine A=adenosine T=thymidine G=guanosine This invention provides a method of determining a chromosomal breakpoint in a subject suffering from multiple myeloma which comprises steps of: obtaining a DNA sample from the subject suffering from multiple myeloma; (b) determining whether there is J and C disjunction in the immunoglobulin heavy chain gene in the obtained DNA sample; obtaining a genomic library having clones which contain genomic DNA fragments from the DNA sample which shows positive J and C disjunction; selecting and isolating clones of the obtained library which show positive hybridization with a probe which is capable of specifically hybridizing with the C but not the J region of the immunoglobulin heavy chain gene; preparing fluorescent probes from the genomic DNA fragments of the isolated clones from step hybridizing said fluorescent probes with metaphase chromosomes; and determining the identity of the chromosomes which are capable of hybridizing to said fluorescent probes, wherein the identification of a chromosome other than chromosome 14 would indicate that the chromosomal breakpoint is between chromosome 14 and the identified chromosome, thereby determining a chromosomal breakpoint in a subject suffering from multiple myeloma.

In an embodiment, step of the above described method of this invention is performed by Southern blotting. In another embodiment, step of the above method of this WO 97/45106 PCT/US97/09065 invention is performed by polymerase chain reaction (PCR) with appropriate probes. Polymerase chain reaction is well known in the art. Since the sequences of both the C and J regions of an immunoglobulin heavy chain gene are known, appropriate probes for PCR may routinely be designed.

In an embodiment, the genomic library is a phage vector library. In another embodiment, the genomic DNA fragments are generated by cleaving genomic DNA from cells of the subject with an appropriate restriction enzyme. In a further embodiment, the restriction enzyme is BamHI. In an embodiment, the restriction enzyme is Sau3AI. In another embodiment, the probe of step is a human IgH J region JH probe. In a further embodiment, the probe of step is a human IgH CL probe. In an embodiment, the probe of step (d) is a human IgH Cy2 probe. In another embodiment, the chromosomal breakpoint identified is a t(6;14) (p25;q32) translocation. In an embodiment, the chromosomal breakpoint identified is a t(l;14) translocation.

This invention provides a method to identify a gene other than the immunoglobulin gene which is located in chromosome 14, altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma which comprises steps of: a) selecting a probe having a sequence of a chromosome other than chromosome 14, identified at the chromosomal breakpoint detected in a subject suffering from multiple myeloma, wherein said probe is capable of hybridizing to the unique sequence of the gene other than the immunoglobulin gene altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma; b) contacting said probe with mRNA isolated from a cell under conditions permitting formation of a complex between said probe and the mRNA; c) isolating the complex resulting from step and d) determining the sequence of the mRNA in the WO 97/45106 PCTUS97/09065 -21isolated complex, thereby determining the identity of the gene.

In an embodiment, step of the method to identify a gene other than the immunoglobulin gene which is located in chromosome 14, altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma comprises steps of: i) synthesizing complementary DNA to the mRNA; and ii) performing sequence analysis of the complementary DNA to determine the sequence of the mRNA.

This invention provides a gene identified by the method to identify a gene other than the immunoglobulin gene which is located in chromosome 14, altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma.

As used herein, "MUM" means any gene rearranged in 14q+ chromosomal abnormalities associated with multiple myeloma.

This invention provides a gene identified by the above method designated MUM-I. This invention provides a gene identified by the above method designated MUM-2.

This invention provides a gene identified by the above method, wherein the gene identified comprises a nucleic acid encoding a MUM protein. In an embodiment, the gene identified by the above method comprises a nucleic acid encoding a MUM-1 protein. In another embodiment, the gene identified by the above method comprises a nucleic acid encoding a MUM-2 protein.

This invention provides an isolated nucleic acid molecule encoding a MUM protein. In an embodiment, the isolated WO 97/45106 PCT/US97/09065 -22nucleic acid molecule encoding a MUM protein is a DNA molecule. In another embodiment, the isolated nucleic acid molecule encoding a MUM protein is a cDNA molecule.

In an embodiment, a cDNA nucleic acid molecule encoding a MUM-1 protein is cloned into a pBluescript KS+ and the resulting plasmid is designated as pcMUMl-1.6a (ATCC Accession No. 97579). Plasmid pcMUM1-1.6a was deposited on May 28, 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Plasmid pcMUM1-1.6a was accorded ATCC Accession Number 97579.

In another embodiment, a partial cDNA nucleic acid molecule encoding a MUM-1 protein is cloned into a pBluescript KS+ and the resulting plasmid is designated as pMUMl-2.4B/N (ATCC Accession No. 97578). Plasmid pMUM1-2.4B/N was deposited on May 28, 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Plasmid pMUM1-2.4B/N was accorded ATCC Accession Number 97578.

In another embodiment, a partial cDNA nucleic acid molecule encoding a MUM-1 protein is cloned into a pBluescript KS+ and the resulting plasmid is designated as pMUM1-7.7B (ATCC Accession No. 97577). Plasmid pMUM1-7.7B was deposited on May 28, 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, WO 97/45106 PCT/US97/09065 -23- U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Plasmid pMUM1-7.7B was accorded ATCC Accession Number 97577.

In another embodiment, a partial cDNA of the nucleic acid molecule encoding a MUM-2 protein is cloned into a pBluescript KS+ and the resulting plasmid is designated as pMUM2-8 (ATCC Accession No. 97580). Plasmid pMUM2-8 was deposited on May 28, 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Plasmid pMUM2-8 was accorded ATCC Accession Number 97580.

In an embodiment, the isolated DNA molecule encoding a MUM protein is a cDNA molecule having the nucleotide sequence shown in Figure 5B-1-5B-3 (SEQ. ID NO 13).

In an embodiment, the isolated DNA molecule encoding a MUM protein is genomic DNA molecule. In an embodiment, the isolated nucleic acid molecule encoding a MUM protein is an RNA molecule.

In an embodiment, the isolated nucleic acid encodes a human MUM-1 protein. In another embodiment, the isolated nucleic acid molecule encodes a human MUM-2 protein.

In an embodiment, isolated nucleic molecule encodes the a human MUM-1 protein having substantially the same amino acid sequence as shown in Figure 5B-1-5B-2 (SEQ. ID NO 14). In WO 97/45106 PCT/US97/09065 -24an embodiment, isolated nucleic molecule encodes a human MUM-1 protein having the same amino acid sequence as shown in Figure 5B-1-B-2 (SEQ. ID NO 14). In an embodiment, the isolated nucleic acid molecule encoding a MUM protein is operatively linked to a promoter of RNA transcription.

This invention provides a vector comprising a cDNA molecule encoding a MUM protein. In an embodiment, a vector comprising cDNA encoding for MUM-1 is designated pcMUM1.6a.

In an embodiment, a vector comprising partial cDNA encoding for MUM-1 is designated pMUM1.2.4B/N. In an embodiment, a vector comprising partial cDNA encoding for MUM-1 is designated pMUMl-7.7B. In an embodiment, a vector comprising partial cDNA encoding for MUM-2 is designated pMUM2-8. In an embodiment, a vector comprises genomic DNA encoding for MUM. In an embodiment, the vector is a plasmid. In an embodiment, a host cell comprises the vector comprising cDNA encoding for MUM. In an embodiment, a host cell comprises the vector comprising genomic DNA encoding for MUM. In a further embodiment, the host cell comprising vectors comprising cDNA encoding for MUM or comprising genomic DNA encoding for MUM is selected from a group consisting of a bacterial cell, a plant cell, and insect cell and a mammalian cell.

This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a MUM protein. This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM protein.

WO 97/45106 PCT/US97/09065 As used herein, the phrase "specifically hybridizing" means the ability of a nucleic acid molecule to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs.

In an embodiment, the nucleic acid probe specifically hybridizes with nucleic acid encoding MUM-I. In an embodiment, the nucleic acid probe is complementary to nucleic acid encoding MUM-i. In an embodiment, the nucleic acid probe specifically hybridizes with nucleic acid encoding MUM-2. In an embodiment, the nucleic acid probe is complementary to nucleic acid encoding MUM-2.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-I is a DNA probe.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-2 is a DNA probe.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-i is a RNA probe.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-2 is a RNA probe.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-I is a genomic DNA probe. In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-2 is a genomic DNA probe.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-i is labeled with a detectable marker. In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid WO 97/45106 PCT[US97/09065 -26encoding MUM-2 is labeled with a detectable marker.

In an embodiment, the detectable marker is selected from the group consisting of a radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-l is linked to a nucleic acid sequence capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule of human chromosome 14. In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-2 is linked to a nucleic acid sequence capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the nucleic acid probe comprises a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM-l protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the nucleic acid probe comprises a nucleic acid molecule of at least 15 nucleotides which is WO 97/45106 PCTIUS97/09065 -27complementary to a sequence of the isolated nucleic acid molecule encoding a MUM-2 protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the nucleic acid probe comprises a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM-1 protein which is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the nucleic acid probe comprises a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM-2 protein which is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the specific break point of the nucleic acid probe comprises a portion of the t(6;14)(p25;q32) translocation. In an embodiment, the specific break point of the nucleic acid probe comprises a portion of a t(l;14) translocation. In an embodiment, the nucleic acid probe comprising a portion of the t(6;14)(p25;q32) translocation is labeled with a detectable marker. In an embodiment, the nucleic acid probe comprising a portion of a t(l;14) translocation is labeled with a detectable marker. In an embodiment, the nucleic acid probe comprising a portion of the t(6;14)(p25;q32) or comprising a portion of a t(l;14) translocation of claim 60, has a detectable marker selected WO 97/45106 PCT/US97/09065 -28from the group consisting of a radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.

This invention provides a method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-1 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-1 protein.

This invention provides a method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-2 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein.

In an embodiment, the rearrangement of nucleic acid encoding MUM-1 protein is detected by contacting the nucleic acid from the sample with a MUM-1 probe under conditions permitting the MUM-1 probe to hybridize with the nucleic acid encoding MUM-1 protein from the sample, thereby detecting the rearrangement of nucleic acid encoding MUM-1 protein in the sample.

In an embodiment, the rearrangement of nucleic acid encoding MUM-2 protein is detected by contacting the nucleic acid from the sample with a MUM-2 probe under conditions permitting the MUM-2 probe to hybridize with the nucleic acid encoding MUM-2 protein from the sample, thereby detecting the rearrangement of nucleic acid encoding MUM-2 protein in the sample.

WO 97/45106 PCTIUS97/09065 -29- In an embodiment, the rearrangement of nucleic acid encoding MUM-i protein is detected by a MUM-I probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-i protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the rearrangement of nucleic acid encoding MUM-2 protein is detected by a the MUM-2 probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-2 protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 1.

In an embodiment, the MUM-i probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-l protein is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the MUM-2 probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-2 protein is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 1.

In an embodiment, the MUM-l probe comprises a specific break point comprising a portion of the t(6;14) (p25;q32) translocation. In an embodiment, the MUM-2 probe comprises a specific break point comprising a portion of a t(l;14) translocation.

WO 97/45106 PCT/US97/09065 In an embodiment, the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-i protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-i protein comprises: a) obtaining DNA from the sample of the subject suffering from multiple myeloma; b) performing a restriction digest of the DNA with a panel of restriction enzymes; c) separating the resulting DNA fragments by size fractionation; d) contacting the resulting DNA fragments with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-i protein, wherein the sequence of a nucleic acid molecule encoding a MUM-i protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 and labeled with a detectable marker; e) detecting labeled bands which have hybridized to the nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-i protein, wherein the sequence of a nucleic acid molecule encoding a MUM-i protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 to create a unique band pattern specific to the DNA of subjects suffering from multiple myeloma; f) preparing DNA obtained from a sample of a subject for diagnosis by steps and g) comparing the detected band pattern specific to the DNA obtained from a sample of subjects suffering from multiple myeloma from step and the DNA obtained from a sample of the subject for diagnosis from step to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.

WO 97/45106 PCT/US97/09065 -31- In an embodiment, the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-2 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein comprises: a) obtaining DNA from the sample of the subject suffering from multiple myeloma; b) performing a restriction digest of the DNA with a panel of restriction enzymes; c) separating the resulting DNA fragments by size fractionation; d) contacting the resulting DNA fragments with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-2 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 and labeled with a detectable marker; e) detecting labeled bands which have hybridized to the nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-2 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 to create a unique band pattern specific to the DNA of subjects suffering from multiple myeloma; f) preparing DNA obtained from a sample of a subject for diagnosis by steps and g) comparing the detected band pattern specific to the DNA obtained from a sample of subjects suffering from multiple myeloma from step and the DNA obtained from a sample of the subject for diagnosis from step to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.

WO 97/45106 PCT/US97/09065 -32- In an embodiment, the size fractionation in step is effected by a polyacrylamide or agarose gel. In an embodiment, the detectable marker is radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.

In an embodiment, the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-1 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-1 protein comprises: a) obtaining RNA from the sample of the subject suffering from multiple myeloma; b) separating the RNA sample by size fractionation; c) contacting the resulting RNA species with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-1 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-1 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 and labeled with a detectable marker; d) detecting labeled bands which have hybridized to the RNA species to create a unique band pattern specific to the RNA of subjects suffering from multiple myeloma; e) preparing RNA obtained from a sample of a subject for diagnosis by steps and f) comparing the detected band pattern specific to the RNA obtained from a sample of subjects suffering from multiple myeloma from step and the RNA obtained from a sample of the subject for diagnosis from step to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.

WO 97/45106 PCTIUS97/09065 -33- In an embodiment, the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-2 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein comprises: a) obtaining RNA from the sample of the subject suffering from multiple myeloma; b) separating the RNA sample by size fractionation; c) contacting the resulting RNA species with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-2 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 15 and labeled with a detectable marker; d) detecting labeled bands which have hybridized to the RNA species to create a unique band pattern specific to the RNA of subjects suffering from multiple myeloma; e) preparing RNA obtained from a sample of a subject for diagnosis by steps and f) comparing the detected band pattern specific to the RNA obtained from a sample of subjects suffering from multiple myeloma from step and the RNA obtained from a sample of the subject for diagnosis from step to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.

In an embodiment, the size fractionation in step is effected by a polyacrylamide or agarose gel. In an embodiment, the detectable marker is radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.

In an embodiment, multiple myeloma associated with the WO 97/45106 PCT/US97/09065 -34expression of a specific human MUM-1 is diagnosed by the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-1 protein in a DNA or RNA sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-1 protein.

In an embodiment, multiple myeloma associated with the expression of a specific human MUM-2 is diagnosed by the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-12 protein in a DNA or RNA sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein.

This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the cDNA molecule encoding a MUM protein. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA molecule encoding a MUM protein. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the RNA molecule encoding a MUM protein.

WO 97/45106 PCT/US97/09065 This invention provides a purified MUM protein. This invention provides a purified MUM-1 protein. This invention provides a purified human MUM-1 protein. This invention provides an antibody directed to a purified MUM-1 protein.

This invention provides an antibody capable of specifically recognizing MUM-1 protein. In an embodiment, the antibody capable of specifically recognizing MUM-1 protein is a human MUM-1 protein.

This invention provides a purified MUM-2 protein. This invention provides a purified human MUM-2 protein. This invention provides an antibody directed to a purified MUM-2 protein. This invention provides an antibody capable of specifically recognizing MUM-2 protein. In an embodiment, the antibody capable of specifically recognizing MUM-2 protein is a human MUM-2 protein.

In an embodiment, the antibody directed to a purified MUM-1 protein is a monoclonal antibody. In an embodiment, the antibody capable of specifically recognizing MUM-1 protein is a monoclonal antibody. In an embodiment, the antibody capable of specifically recognizing MUM-1 protein is a human MUM-1 protein.

In an embodiment, the antibody directed to a purified MUM-2 protein is a monoclonal antibody. In an embodiment, the antibody capable of specifically recognizing MUM-2 protein is a monoclonal antibody. In an embodiment, the antibody capable of specifically recognizing MUM-2 protein is a human MUM-2 protein.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a WO 97/45106 PCT/US97/09065 -36sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-1 protein so as to prevent overexpression of the mRNA molecule effective to prevent overexpression of a human MUM-1 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to a cDNA molecule encoding a MUM protein effective to prevent overexpression of a human MUM-1 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to a genomic DNA molecule effective to prevent overexpression of a human MUM-1 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-2 protein so as to prevent overexpression of the mRNA molecule effective to prevent overexpression of a human MUM-2 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a 37 sequence capable of specifically hybridizing to a cDNA molecule encoding a MUM protein effective to prevent overexpression of a human MUM-2 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to a genomic DNA molecule effective to prevent overexpression of a human MUM-2 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific °methods and results discussed are merely illustrative of the invention as 15 described more fully in the claims which follow thereafter.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

WO 97/45106 PCT/US97/09065 -38- EXPERIMENTAL DETAILS Materials and Methods Cell lines. The following myeloma cell lines were used in the present study: SK-MM-1, RPMI-8226, U266, EJM, XG-1, XG-2, XG-4, XG-5, XG-6, XG-7, and XG-10. The RPMI-8226 cell line was obtained through the American Type Culture Collection (ATCC, Rockville, MD). SKMM-1 and U-266 cell lines were gifts from Dr. A. N. Houghton and Dr. K. Nilsson, respectively (18; 12). Characterization of these cell lines were previously reported. Six XG cell lines were gifts from Dr. B. Klein and were cultured in RPMI 1640 containing fetal calf serum (FCS), SxlO-smol/L 2-ME, and rIL- 6(lng/mL)(13;19). Other myeloma cell lines used were all IL-6 independent. The SK-MM-1 cell line was used to isolate the chromosomal breakpoint carrying the 14q+ chromosome without any information on the donor chromosome. XG-1, XG-2, XG-6, XG-8 cell lines are reported to carry the t(11;14) (ql3;q32) translocation. XG-5 cells also share both t(ll;14) and t(8;14)(q24;q32).

Southern and Northern blot analyses. Southern blot analysis was performed as previously described Briefly, ten micrograms of high molecular-weight DNA extracted from each cell line was digested to completion with BamHI and HindIII restriction enzymes, size- fractionated on 0.7% agarose gel, and transferred onto Duralose nitrocellulose membrane (Stratagene) according to the manufacturer's instructions.

Blots were hybridized with a random-primed DNA probe and washed at 60 0 C in 0.2 x SSC and 0.1 SDS for 5 minutes.

Genomic probes used in this study were as follows; human IgH WO 97/45106 PCT/US97/09065 -39- J region JH probe (6.6kb BamHI-HindIII fragment) was provided by Dr. J. V. Ravetch, human IgH Cg probe (1.3 kb EcoRI fragment) was provided by Dr. S.J. Korsmeyer. Human IgH region Cy 2 probe was provided by Dr. C. Croce.

Northern blot analysis was performed as described previously Briefly, a 10 pg aliquot of total RNA was loaded on each lane and probed with a 2.1H probe of the MUMI gene (Figure 2A). GAPDH or p-actin probes were used as controls for amount of total RNA.

Genomic library. High molecular-weight DNA of SK-MM-1 cell line was digested completely with BamHI and partially with Sau3AI, and size-fractionated by using a low-melting point agarose gel. DNA ranging from 10kb to 23kb were purified and ligated into the BamHI sites of X-DASH II phage vector (Stratagene, La Jolla, CA). After packaging, 3 x 105 and 6 x 10 5 recombinant clones of the BamHI digested library and partially digested library were screened with JH and C probes, respectively. To isolate the germline region of the 6p25 locus, a commercially available human placental library (Stratagene) was screened. Positive clones were mapped with restriction enzymes by partial digestion of the phage DNAs followed by probing with T7 and T3 primers labeled with T4 polynucleokinase and 32p-yATP.

cDNA library. A phage library constructed by oligo-dT and random-priming normal human spleen RNA (Clontech) was screened by 2.1H probe (Figure 2A) to isolate initial MUM1 cDNA clones. After the first round of screening, positive clones were used as probes to walk to the 5' side using the same library. Positive clones were subcloned into WO 97/45106 PCT/US97/09065 pBluescript and analyzed for mapping and sequencing.

DNA sequencing. DNA sequences were determined by the dideoxy chain termination method and analyzed by an ABI(Applied Biosystems) autosequencer. Deletion mutants for sequencing were prepared using exonuclease III and mung bean nuclease. cDNA sequences were analyzed with the Genetics Computer Group (GCG) programs. Sequence homology searches were carried out through the BLAST E-mail server at the National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD.

Fluorescence in situ hybridization (FISH). Metaphase chromosome from human lymphocytes were prepared. A biotin-labeled probe was prepared by nick-translation using Bio-16-dUTP. Conditions for hybridization and washing were described previously (22).

Experimental Results IgH gene rearrangement of the SK-MM-I cell line. In BamHI digestion, the JH probe detects two rearranged bands of the size of 12.0 kb and 9.7kb (Fig The 9.7 kb band is comigrated with that probed with Cy2 probe, suggesting it to be a physiological rearrangement, although this cell line secretes only X chain. One allele of the Cp locus is deleted and another is rearranged (6.5 kb) without being comigrated with rearranged bands of JH. Hybridization with a Ca probe showed only the germline band (data not shown).

These results suggested the possibility of the chromosomal breakpoint between JH and Cp locus. Hence, the 12.0 kb and the 6.5 kb bands detected by JH and Cu were considered to ,4 WO 97/45106 PCT/US97/09065 -41represent unknown derivative chromosome and derivative 14 chromosome, respectively.

Molecular cloning of the t(6;14)(p25;q32) breakpoint.

A

genomic library constructed with BamHI complete digestion was screened with a JH probe to isolate the 12.0 kb BamHI band. Another library constructed with Sau3AI partial digestion was screened with a Cp probe to isolate phage clones containing the 6.5 kb BamHI fragment. Two phage clones, XSKB-4a and X SKS-3, considered to represent the unknown derivative and derivative 14 chromosomes respectively, were obtained (Fig 2A). A 0.7 kb BamHI-HindIII probe (0.7B/H) of the XSKS-3 was used to confirm the comigration with the rearranged 6.5 kb Cp band by Southern analysis (Fig The chromosomal origin of the centromeric side of the XSKB-4a and telomeric side of the ASKS-3 were confirmed by hybridization to a somatic cell hybrid DNA panel with a 4.5 kb ApaI fragment(4.5A) and 2.1 kb HindIII(2.1H) probes. Both probes showed positive signals in hybrid cell DNA containing a human chromosome 6 (data not shown). These probes were also used to isolate the germline chromosome 6 region by screening the human placental genomic library. One of the phage clone DNA (XMUM-3) was used as a probe for FISH analysis. It identified the localization of this region to be chromosome 6 short arm p25 (Fig To investigate the precise breakpoint within the IgH gene, a 1.5 kb HindIII-EcoRI fragment of the XSKS-3, containing the breakpoint on derivative 14 chromosome was sequenced. The breakpoint was confirmed to be just 3' to the switch p(S p) repetitive sequences [SEQ ID NO: 15] (Fig 2B). Nucleotide sequencing of the region around the breakpoints of chromosome 6 and WO 97/45106 PCT/US97/09065 -42derivative 6 chromosome showed that the chromosomal translocation was reciprocal with minimum deletion of both the IgH and 6p25 sequences.

Transcriptional unit in the vicinity of the 6p25 breakpoint.

An attempt to find a functional transcriptional unit in the vicinity of the breakpoints was made. Although a 4.5A probe on derivative 6 chromosome could not detect any transcripts, a 2.1H probe on derivative 14 chromosome detected a single 6 kb transcript in the SK-MM-1 cell line. Accordingly, this gene was designated as MUM1 (multiple myeloma oncogene 1).

The same probe was used to study the expression of the MUM1 gene in various hematopoietic cell lines. The 6 kb message was expressed at high levels in most B cell lines and at low levels in peripheral T cell lines (Fig 4A). Cell lines derived from immature T cells, the myelomonocytic lineage, and erythroid lineage do not seem to express MUM1. In B cells, MUM1 appears to be expressed throughout the development from the preB cell stage to the plasma cell stage (Fig 4B). However, some of the Burkitt's lymphoma derived cell lines such as BJA-B did not express this gene (data not shown). The expression level of the MUM1 transcript in myeloma cell lines was also examined (Fig 4C).

The SK-MM-1 cell line showed a 7.5-fold overexpression when compared with the other three IL-6 independent cell lines, suggesting a deregulated expression of the translocated allele. It is of interest that the IL-6 dependent XG-4, XG-7, and XG-10 cell lines are also expressing at high levels. Particularly, expression in the XG-7 cell line is 19.9 times the average of the aforementioned control cell lines.

WO 97/45106 PCT/US97/09065 -43- MUMI cDNA cloning, sequencing, and homology search.

Human spleen cDNA library was initially screened with a 2.1H probe followed by three times walking to 5' side using cDNA probes. A 5.5kb cDNA, approximately corresponding to the size detected by Northern analysis was isolated. This cDNA contained a 1,353 base pair open reading frame (ORF) and a long 3' untranslated region (Fig 5A). The ORF encodes for a protein of 451 amino acids with a predicted molecular weight of 50 kD (Fig 5B-1-B-2; SEQ ID NO:14). The putative ATG initiation codon at position 217 has G at the -3 position which corresponds to the Kozak consensus sequence The ORF is preceded by two in-frame stop codons. A database search demonstrated a significant similarity between MUM-1 ORF and the interferon regulatory factor (IRF) family proteins. The NH 2 -terminal of the MUM-1 ORF (SEQ ID NO:1) shares a high homology with all of the IRF family proteins (SEQ ID NOS: 2-7) which share a characteristic DNA binding motif consisting of the conserved tryptophan residues (Fig 6A). The COOH-terminal (SEQ ID NO:8) also has a high homology with ICSBP (SEQ ID consensus sequence binding protein)(21), ISGF3y (SEQ ID NO:11) (interferon-stimulated gene factor-3 gamma)(22), and IRF-3 protein (SEQ ID NO:12)(23)(Fig 6B), although it did not have any homologous regions with IRF-1 and IRF-2 protein. The highest similarity and identity were found with a possible mouse homolog, LSIRF (SEQ ID NOS:2 and 9) (lymphoid specific interferon regulatory factor)/Pip (PU-1 cofactor protein-1)(24,25).

A

high similarity was found with ICSBP ISGF3y and IRF3 among the human IRF family protein members. A gene sequence encoding a nearly identical protein was recently deposited in GenBank. This gene, termed WO 97/45106 PCT/US97/09065 -44- ICSAT (interferon consensus sequence binding protein in adult T-cell leukemia cell lines or activated T cells) is likely to be the same gene as MUM1 (26).

Breakpoints at MUM1 locus in multiple myeloma.

In order to analyze the exact location of the SK-MM-1 breakpoint at the 6 p 25 locus and to explore the frequency of the MUM1 gene involvement in myeloma cases, we walked nearly in a human placental genomic phage library around the MUM1 gene and determined the rough exon-intron structure as shown in Figure 7 (Fig. The SK-MM-1 breakpoint was located 3' to the last exon, containing a poly A additional signal, consistent with an unaltered size of the MUM1 transcript of this cell line in Northern analysis. Seven repeat-free genomic probes shown in Figure 7 have been used to investigate the rearrangement in Southern analyses of the 11 MM cell lines and 18 MM cases. One case (case displayed rearranged bands in BamHI and XbaI digests when analyzed using a 0.9A probe located at 3' to the MUM1 gene.

Cloning of the MUM2 locus from the U-266 multiple myeloma cell line.

Using an experimental strategy analogous to the one described for the cloning of the MUM1 gene from the SK-MM-1 cell line, a second genetic locus altered in multiple myeloma (MUM2) was identified by analyzing the U-266 multiple myeloma cell line. Briefly, Southern blot analysis using BamH restiction digestion and various Ig probes showed that U-266 DNA contained two rearranged fragments (shown by arrowheads in Fig. 9) containing Ca sequences and lacking J sequences. These two fragments (der 14 and 14q32 in Fig. 9) were cloned from a genomic library constructed from U-266 WO 97/45106 PCT/US97/09065 DNA along with a normal 14q 32 locus (14q32 germline in Fig.

In order to determine whether a gene was located in proximity to the chromosomal breakpoints in der 14, the BE restriction fragment (see Fig. which was at the opposite side of the Ig Ca sequences, was used to probe a Northern blot carrying RNA from various MM cell lines. The results (Fig. 10) showed that a 1.9 kb mRNA was detectable in some of these cell lines including U-266. This result showed that a gene, called MUM2, normally not present within the Ig locus on chromosome 14q32, had been translocated in proximity of the Ig locus in U-266 cells. Since the Ig locus contains strong transcriptional regulatory elements, it is likely that the expression of this gene is deregulated in these cells. The structure of the MUM2 gene and its protein are currently under investigation. The 2.5 BE probe and other probes derived from the der 14 phage can be used to screen MM cases for MUM2 rearrangements as shown for MUM1 (Fig. 7).

Experimental Discussion Using the experimental strategies used for the identification of the MUM1 and MUM2 genes in the SK-MM-1 and U-266 cell lines, respectively, it is possible to analyze most MM cases and isolate the corresponding genes. The scheme shown in Fig. 11 shows that the physiological IgH gene rearrangements (Fig. 11A) typically maintain linkage of C and J sequences and this linkage becomes detectable by using an appropriate restriction enzyme digestion (BamHI in the example in Fig. 11). Conversely, chromosomal translocations (14q+) affecting the IgH locus on 14q32 lead to breakage of the C-J linkage and the two sets of sequences WO 97/45106 PCT/US97/09065 -46appear on distinct restriction fragments. (Fig. 11B) Table 1 shows the application of this analysis to a panel of MM cell lines and biopsies. The results show that at least of cases show breakage of the C-J linkage within Ig J or switch regions. The restiction fragments containing either C or J sequences (R in Table 1) can be cloned as shown for the SK-MM-l and U-266 cell lines and the genes flanking the chromosomal breakpoints can be used as probes to screen additional MM cases for similar rearrangements, whereas the sequence of the genes can be used to understand the consequences of these genetic lesions in multiple myeloma.

Cloning of the chromosomal breakpoints and corresponding genes is currently ongoing for all of the MM cases shown in Table 1.

The method of analysis of 14q+ chromosomal translocations and identification of the genes altered in multiple myeloma of this invention will allow 1) the determination of chromosomal sequences involved in 14q+ translocations, the most important cytogenetic lesion associated with MM pathogenesis elucidation; 2) elucidation of specific gene lesions for MM; 3) a diagnostic method based on gene/DNA lesion and 4) a therapeutic approach aimed at counteracting the action of abnormal gene products.

WO 97/45106 WO 9745106PCT[US97/09065 -47- Table 1. Summary of JH-C breakage analysis in MM cell lines and biopsies (cases).

Rearrangement involving physiologic Ig recombinations, i.e. retaining JH-C linkage are marked as rearrangements lacking JH-C linkage, and therefore suggesting a 1 4q+ chromosomal breakpoint, are marked as R. The latter represents candidates for cloning an further analysis.

Cell Line/Case slg JH Cg. Ca~ Sy 3 possible breakpoint locus RPMI-8226 U-266

EJM

XG-l XG-2 XG-4 XG-6 XG-7

X

EX~

GX

AK,

GX

GK,

GX

AK

R*IR

D/D

R/D

RJR*

R*ID

R*IR

R/D

R*IR

RID

DID

R/D

DID

D/D

DID

D/D

G

R/R/G

G

R*

G

RIG

R*IG

RIG

G

R*IG

G

R*IG

G

R*IG

RID

S a JH-S~t

ND

JHS

Sy SK-MM- 1 JH-Sgt CASE125 R* G G R* ND CASE33 RIR* G G RIR* S Y CASE34 R* G R* G ND CASE93 R* G R* G ND CASE91 R* R* R G S a CASE128 R* G R* G ND comigrated bands with JH; it, target bands to isolate; ND, not determined WO 97/45106 PCT/US97/09065 -48- Possible breakage in switch regions: Cell Lines 4/11(36%) Cases 2/6 (33%) Possible breakage in JH switch regions: Cell Lines 9/11(82%) Cases 2/6 (33%) Total 6/17 Total 11/17 WO 97/45106 PCT/US97/09065 -49- References for First Series of Experiments 1. Rabbitts, T.H. Chromosomal translocations in human cancer. Nature 372:143-149, 1994.

2. Dalla-Favera, Bregni, Erickson, Patterson, Gallo, Croce, C.M. Human c-myc oncogene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Nat Acad Sci USA 79:7824, 1982.

3. Tsujimoto, Jaffe, Cossman, Gorham, J., Nowell, Croce, C.M. Clustering of breakpoints on chromosome 11 in human B-cell neoplasms with the t(ll;14) chromosome translocation. Nature 315:340-343, 1985.

4. Motokura, Bloom, Kim, Juppner, H., Ruderman, Kronenberg, Arnold, A. A novel cyclin encoded by a bcl-1 linked candidate oncogene.

Nature 350:512-515, 1991.

Tsujimoto, Yunis, Onorato-Showe, L., Erikson, Nowell, Croce, C.M. Molecular cloning of chromosomal breakpoint of B-cell lymphomas and leukemias with the t(ll;14) chromosome translocation. Science 224:1403-1406, 1984.

6. Cleary, Sklar, J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally active locus on chromosome 18. Proc WO 97/45106 PCT/US97/09065 Natl Acad Sci USA 82: 7439, 1985.

7. Bakhshi, Jensen, Goldman, Wright, J.J., McBride, Epstein, Korsmeyer, S.J. Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 41:889, 1985.

8. Ye, Lista, Lo Coco, Knowles, Offit, Chaganti, Dalla-Favera, R. Alterations of a zinc finger-encoding gene, BCL-6, in diffuse large-cell lymphoma. Science 262:747-750, 1993.

9. Dewald, Kyle, Hicks, Greipp, P.R. The clinical significance of cytogenetic studies in 100 patients with multiple myeloma, plasma cell leukemia, or amyloidosis. Blood 66:380-390, 1985.

Gould, Alexanian, Goodacre, Pathak, S., Hecht, Barlogie, B. Plasma cell karyotype in multiple myeloma. Blood 71:453-456, 1988.

11. Weh, Gutensohn, Selbach, Kruse, R., Wacker-Backhaus, Seeger, Fiedler, Fett, Hossfeld, D.K. Karyotype in multiple myeloma and plasma cell leukemia. Eur J Cancer 29A:1269-1273, 1993.

12. Jernberg, Zech, Nilsson, K. Cytogenetic studies on human myeloma cell lines. Int J Cancer 811-817, 1987.

WO 97/45106 PCT/US97/09065 -51- 13. Zhang, Gaillard, Robillard, Lu, Gu, Jourdan Boiron, Bataille, Klein, B. Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma. Blood 83:3654-3663, 1994.

14. Seto, Yamamoto, lida, Akao, Utsumi, Kubonishi, Miyoshi, Ohtsuki, T., Yawata, Namba, Motokura, Arnold, A., Takahashi, Ueda, R. Gene rearrangement and overexpression of PRAD1 in lymphoid malignancy with t(11;14) (q13;q32) translocation. Oncogene 7:1401 -1406, 1992.

Rabbitts, Douglas, Fisher, Nacheva, Karpas, Catovsky, Melo, Baer, R., Stinson, Rabbitts, T.H. Chromosome abnormalities at llq13 in B cell tumours. Oncogene 3:99103, 1988.

16. Fiedler, Weh Hossfeld, D.K. Comparison of chromosome analysis and BCL-1 rearrangement in a series of patients with multiple myeloma. Br J Haematol 81: 58-61, 1992.

17. Taniwaki, Nishida, Takashima, Nakagawa, Fujii, Tamaki, Shimazaki, Horiike, Misawa, Kashima, K. Nonrandom chromosomal rearrangements of 14q32.3 and 19p13.3 and preferential deletion of lp in 21 patients with multiple myeloma and plasma cell leukemia. Blood WO 97/45106 PCT/US97/09065 -52- 84: 2283-2290, 1994.

18. Sun, Kitchingman, G.R. Sequencing of selected regions of the human immunoglobulin heavy-chain gene locus that completes the sequence from JH through delta constant region. DNA sequence 1:347-355, 1991.

19. Eton, Scheinberg, Houghton, A.N.

Establishment and characterization of two human myeloma cell lines secreting kappa light chains.

Leukemia 3: 729-735, 1989.

Mazars, Portier, Zhang, Jourdan, Bataille, Theillet, Klein, B. Mutations of the pS3 gene in human myeloma cell lines.

Oncogene 7: 1015-1018, 1992.

21. lida, Seto Yamamoto, Tojo, Asano, Kamada, Ariyoshi, Takahashi, Ueda, R. MLLT3 gene on 9p22 involved in t(9;11) leukemia encodes a serine/proline rich protein homologous to MLLT1 on 19pl3. Oncogene 8(11):3085-3095, 1993.

22. Rao, Murty, Gaidano, G., Hauptschein, Dalla-Favera, and Chaganti, R.S.K. Subregional mapping of 8 single copy loci to chromosome 6 by fluorescence in situ hybridization.

Cytogenet. Cell Genet. 66:272-273, 1994.

23. Kozak, M. The scanning model for translation: an update. J. Cell. Biol. 108:229-241, 1989.

WO 97/45106 PCT/US97/09065 -53- 24. Driggers, Ennist, Gleason, Mak, Marka, Levi, Flanagan, J.R., Appella, Ozato, K. Proc Natl Acad Sci USA 87: 3743-3747, 1990.

Veals, Schindler, Leonardo, Fu, X-Y., Aebersold, Damell, Levy, D.E. Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and myb families of DNA-binding proteins. Mol Cell Biol 12: 3315-3324, 1992.

26. Grant, Vasa, Deeley, R.G. cIRF-3, a new member of the interferon regulatory factor(IRF) family that is rapidly and transiently induced by dsRNA. Nucleic Acid Res 23:2137-2145, 1995.

27. Matsuyama, Grossman, Mittrucker, H-W., Siderovski, Kiefer, Kawakami, T., Richardson, Taniguchi, Yoshinaga, S.K., Mak, T.W. Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element(ISRE).

Nucleic Acid Res 23:2127-2136, 1995.

28. Eisenbeis, Singh, Storb, U. Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev 9:1377-1387, 1995.

29. Yamagata, Nishida, Tanaka, Sakai, R., WO 97/45106 PCT/US97/09065 -54- Mitani, Yoshida, Taniguchi, Yazaki, Y., Hirai, H. A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes. Mol Cell Biol 16:1283-1294, 1996.

WO 97/45106 PCT/US97/09065 Second Series of Experiments The pathogenesis of multiple myeloma an incurable tumor affecting terminally differentiated B cells, is unknown Chromosomal translocation (14q,) affecting 14q32 and unidentified partner chromosome(s) are common in this tumor suggesting that they may cause the activation of novel oncogenes. By cloning and mapping the chromosomal breakpoints in a MM cell line, this study shows that the 14q+ translocation represents a t(6;14)(p25;q32) and that this aberration is recurrent in MM since it was found in 2 of 11 MM cell lines. The translocation juxtaposes the immunoglobulin heavy-chain (IgH) locus to the MUM1 (multiple myeloma oncogene 1)/IRF4 gene, a member of the interferon regulatory factor (IRF) family involved in the control of B cell proliferation and differentiation. As a result, the MUM1/IRF4 gene is overexpressed, an event that may contribute to tumorigenesis since we show that MUM1/IRF4 has oncogenic activity in vitro. These findings identify a novel genetic alteration associated with MM with implications for the pathogenesis and diagnostics of this tumor.

Analysis of MM karyotypes by fluorescence in situ hybridization (FISH) using probes from the IgH locus have shown that chromosomal translocations involving the IgH locus on band 14q32 (14q+) are common in MM (62% of cases) A recent study has shown that most 14q+ chromosomes represent translocations of a variety of loci into the IgH switch region but except for a fraction of cases involving the BCL-I locus on chromosome 11q13 the genes involved in the partner chromosomes have not been identified. In order to investigate the nature of the sequences linked to the IgH locus in 14q+ chromosomes, a number of cell lines were screened for the presence of WO 97/45106 PCT/US97/09065 -56abnormally rearranged IgH loci which could reflect the presence of chromosomal translocations. A Southern blot hybridization assay was used aimed at distinguishing rearranged IgH alleles containing IgH J regions (JH) linked to C regions (representing physiologically rearranged IgH loci) from those in which JH and C sequences are not linked, as these alleles reflect illegitimate IgH switch recombinations often representing chromosomal translocations. Using this assay, 9 of 11 MM lines tested showed evidence of illegitimate switch recombinations (not shown).

Among these cell lines, the pattern of IgH gene rearrangements detectable in the SK-MM-1 strongly suggested the presence of a chromosomal translocation (Fig. 14A).

BamHI digestion of SK-MM-1 genomic DNA and hybridization with JH probe showed two rearranged restriction fragments (12.0 and 9.7kb, Fig. 14A). One (9.7 kb) was hybridized also with a Cy2 probe, suggesting it to be derivation from a physiological rearrangement, while the second (12.0 kb) was not hybridized with any C probe. Conversely, hybridization with a Ci probe led to the identification of a 6.5 kb fragment which did not contain any J sequences.

Hence, the 12.0 kb and 6.5 kb bands detected by JH and CA respectively were considered to represent potential chromosomal breakpoints and cloned from a phage library constructed from SK-MM-1 genomic DNA. Two phage clones (XSKB-4a; XSKS-3; Fig. 14B) were representative of the two rearranged fragments detected in SK-MM-1 DNA in that they displayed BamHI restriction fragments containing JH and CA sequences not reciprocally linked as well as sequences which based on their restriction map could not derive from the IgH locus. A 0.7B/H probe from the XSKS-3 phage insert representing non-IgH sequences (Fig. 14B) hybridized to a BamHI fragment comigrating with that containing Cp sequences WO 97/45106 PCT/US97/09065 -57in SK-MM-1 DNA, indicating its derivation from the abnormally rearranged Cg fragment (Fig. 14A). The same probe was used to clone its corresponding normal locus from a library constructed from normal human DNA (Fig. 14B).

This locus was assigned the name MUM1 (multiple myeloma oncogene 1) by the Nomenclature committee of the Genome Data Base for the gene locus in humans.

The MUM1 locus was mapped to chromosome 6 by hybridization of the 2.1H and 4.5A probes to a somatic cell hybrid panel representative of individual human chromosomes (not shown).

Using a 360kb non-chimeric YAC (y927E3) spanning the MUM1 region, the MUM1 locus was further submapped to 6p25 by FISH analysis (Fig. 15A-B). The same YAC probe hybridized to the 14q+ chromosome as well as to a der(6) chromosome (as well as to normal chromosomes 6) in SK-MM-1 cells (not shown) as well in a second MM cell line (XG-7) (Fig. 15C-D), while no abnormality was detectable in additional 9 MM lines. Taken together, these observations indicated that the 14q+ chromosome present in SK-MM-1 and XG-7 cells was part of a reciprocal t(6;14)(p25;q32) translocation. This abnormality is recurrent in MM since it was detectable in 2 of 11 cases tested.

Next it was investigated whether the MUMI locus adjacent to the chromosomal breakpoints contained a transcriptional unit. Probe 2.1H detected a major 6 kb RNA in B-cell lines representative of various stages of B cell differentiation (from preB cells through to plasma cells), as well as in lines displaying a mature T cell phenotype (Fig. 16A-B).

This result indicates that 6p25 sequences immediately adjacent to the chromosomal breakpoints are part of a gene (MUMI) which is transcribed in lymphoid tissues.

WO 97/45106 PCT/US97/09065 -58- The corresponding cDNA was cloned from a cDNA library derived from normal human spleen library. Nucleotide sequence analysis of clones spanning 5.5 kb MUM1 cDNA sequences showed an open reading frame (ORF) encoding for 451 amino acids with a predicted molecular weight of 50 kD (data not shown). An homology search in the human gene data bank revealed that MUM1 was virtually identical to the IRF4 gene a member of IRF family of transcription factors.

The MUM1/IRF4 cDNA was used to determine the exon-intron organization of the corresponding genomic locus (Fig. 14B).

This allowed to determine that the chromosomal breakpoint was located 3' to the MUM1/IRF4 gene in SK-MM-1 cells (Fig.

14B). Probes spanning 55kb of the MUMI locus including the entire MUM1/IRF4 gene failed to detect rearrangements in 11 MM cell lines and 18 MM cases studied (not shown), including the XG-7 lines which was shown to contain a t(6;14)(p25;q32) by FISH analysis. These results indicate that, analogous to other translocations involving Ig genes, the breakpoints of t(6;14)(p25;q32) can scatter along 6p25 at variable distance from the MUM2 locus.

To investigate the consequences of chromosomal translocation on MUMI/IRF4 expression, comparisons were made of the levels of MUM1/IRF4 RNA in MM cell lines carrying t(6;14) (p25;q32) (SK-MM-1, XG-7) or lacking detectable 6p25 abnormalities.

In general, the levels of MUM1/IRF4 RNA expression tended to be higher (3.4-fold) in interleukin-6 (IL-6) dependent than in IL-6 independent MM cell lines (Fig. 16C-D). However, SK-MM-1 and the XG-7 cell line displayed the highest levels of MUM1/IRF4 expression, a 7.5- and a 6-fold overexpression compared to the average of other MM cell lines (Fig. 16C-D).

To study whether in the same cell lines increased MUM1/IRF4 RNA expression was associated with increased levels of WO 97/45106 PCT/US97/09065 -59protein expression, analysis was made of the levels of MUM1/IRF4 specifically bound to DNA by electrophoretic mobility shift analysis (EMSA) of nuclear extracts using a IRF binding site as a probe (GBP-ISRE). GBP-IRSE-bound MUM1/IRF4 could not be detected by EMSA in any of the cell lines tested as it is occasionally the case with other transcription factors which form unstable complexes with DNA in vitro; however, these complexes could be stabilized by the addition of anti- MUM-1/IRF-4 antibodies (but not by control antibodies) and became visible as "supershift" bands (Fig. 16E). Both cell lines carrying alterations of the MUM1 locus (SK-MM-1 and XG-7) showed higher amounts (3 to of DNA-bound MUM1/IRF4 than control MM lines, suggesting that overexpression of the MUMI/IRF4 gene was associated with increased amounts of functional MUM1/IRF4 protein.

To investigate whether MUM1/IRF4 overexpression could contribute to malignant transformation, it was tested whether transfection of a MUM1/IRF4 expression vector in Rat-1 fibroblasts could increase their clonogenic properties in agar, a typical sign of malignant transformation. The oncogenic properties on cellular growth was studied. Rat-1 cells were transfected with an expression vector (CMV-MUM1-HA) in which a CMV promoter drives the expression of MUM1/IRF4 tagged by a hemagglutinin (HA) epitope recognizable by a specific monoclonal antibody. Fig. 17 shows that CMV-MUM1-HA transfected Rat-1 clones and expressing detectable levels of exogenous MUM1-HA protein (Fig. 17A), but not control (CMV) transfected clones, acquired anchorage independent growth in soft agar (Fig.

17A-B). These results indicate that MUM1/IRF4 can behave as an oncogene in vitro and support the notion that it may contribute to oncogenesis in vivo.

WO 97/45106 PCT/US97/09065 Chromosomal translocations involving the MUM1/IRF4 gene represent the first lesion specifically associated with MM.

Several studies have shown that gene alterations commonly associated with most human tumors, such as inactivation of the p53 tumor suppressor gene and RAS oncogene mutations, can be found in MM (11,12). However, these lesions are found at low frequency and in association with advanced stages of the disease, suggesting that their role may be limited to tumor progression. The association with 14q+, a cytogenetic aberration typical of lymphoid malignancies and often detectable at diagnosis suggest that MUM1/IRF4 deregulation may be specifically associated with early stages of MM development.

A role of MUM1/IRF4 deregulation in oncogenesis is supported by its oncogenic activity in vitro (Fig. 17) as well as by a number of observations on the biological function of IRFs Various members of this family of transcription factors have been shown to be involved in the control of cell proliferation, differentiation and apoptosis in response to cytokines such as interferon (IFN) and IL-6 (15-18). Some members of the IRF family of genes have been directly implicated in the pathogenesis of various tumors, including ICSBP, whose loss is associated with chronic myelogeneous leukemia (CML) (19-21). Targeted disruption in the mouse germ-line has shown that IRF4 itself controls the differentiation of B cells into plasma cells Since its function does not appear to be regulated by IFN MUM1/IRF4 may act as an effector of other cytokines which regulate B cell differentiation. Interestingly, MUM1/IRF4 gene expression appears to be regulated by IL-6 (Fig. 16 and data not shown), suggesting that its deregulated expression may contribute to the IL-6 autocrine loop that has been shown to support MM cell growth (23).

*F

WO 97/45106 PCT/US97/09065 -61- The observation that the MUM1 locus is involved in a subset, but not all 14q+ alterations underscores the biological heterogeneity of MM. This heterogeneity may correlate with the heterogeneous clinical behaviour of MM. FISH analysis of MM cases using probes for the MUM1 locus may be useful to investigate the frequency of MUM1 alterations in large panels of MM cases and to determine whether this lesion identifies a prognostically distinct subset of tumors.

EXPERIMENTAL DETAILS Methods Cell lines and patient samples. The MM cell lines used in this study are as follows: SK-MM-1, RPMI-8226, U-266, EJM, XG-1, XG-2, XG-4, XG-5, XG-6, XG-7, and XG-10. RPMI-8226 cell line was obtained through American Type Culture Collection (ATCC, Rockville, MD). SK-MM-1 (24) and U-266 cell lines are gifts from Dr. A. N. Houghton (Memorial Sloan-Kettering Cancer Center, New York, NY) and Dr. K.

Nilsson (University of Uppsala, Uppsala, Sweden), respectively. Six XG cell lines were cultured in the presence of Ing/mL recombinant IL-6 (rIL-6) as described previously (26).

Southern and Northern blot analyses. Southern and Northern blot analyses were performed as described previously (27).

Genomic probes used in this study were as follows; human IgH JH probe (6.6kb BamHI-HindIII) and IgH CA probe (1.3 kb EcoRI) were provided by Dr. S. J. Korsmeyer (Washington University, St Louis, MO). Human IgH Cy2 probe (4.0 kb HindIII-BamHI) was provided by Dr. V. Bertness (National Cancer Institute, Bethesda, MD).

Cloning breakpoints, cDNA library screening and DNA WO 97/45106 PCT[US97/09065 -62sequencing. Genomic libraries from SK-MM-1 and human placental DNAs were constructed as described previously Oligo-dT and random-primed human spleen library (Clontech, Palo Alto, CA) was screened by 2.1H probe (Fig.

14B) to isolate initial MUM1 cDNA clones. After the first round of screening, positive clones were used as probes for walking. Positive clones were subcloned into pBluescript and analyzed for mapping and sequencing. DNA sequences were performed as described previously Sequence homology searches were carried out through the BLAST E-mail server at the National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD.

FISH and isolation of MUM1 YAC. Preparation of metaphase spreads and detailed procedure of FISH and Yeast Artificial Chromosome (YAC) DNA extraction were reported previously PCR primers (sense 5'-TACTCGCACCTCTTGGCT-3'; antisense 5'-CTGGAGAGCAATGAACGG-3') derived from the 6p25 sequence within the last exon of the MUM1 gene were used to screen a human CEPH YAC DNA pools Research Genetics, Huntsville,

AL).

Electrophoretic mobility shift assay (EMSA). Preparation of nuclear extract, EMSA and antibody-mediated supershift assays were performed as described GBP-ISRE sequence used as a probe is 5'-AAGTACTTTCAGTTTCATATT-3' Binding buffer used in EMSA was 10mM Tris-HCl (pH 50mM KC1, ImM dithiothreitol, ImM EDTA, 0.1% Triton X-100 and 12.5% glycerol. Antiserum against ICSAT/MUM1 was a gift from Drs. T. Yamagata and H. Hirai (Tokyo University, Tokyo, Japan).

Expression constructs. The MUM1 cDNA (nt.l-1,461) encoding a full length ORF was connected in-frame to the COOH-terminal HA (MAYPYDVPDYASLGPGP) tag, blunt-ended by WO 97/45106 PCT/US97/09065 -63- Klenow enzyme, and cloned into blunt-ended Not I site of pHeBo-CMV eukaryotic expression vector.

Western blot analysis. Cell pellets prepared from MM and Rat-1 cells were resuspended in 1 x Laemmli sample buffer and boiled for 5 minutes. Protein lysates derived from 106 cells were fractionated on an SDS 12.5% acrylamide gel and transferred to a nitrocellulose membrane (Schleicher Schuell, Keene, NH). The filter blocked with 5% milk in Tris-buffered saline (TBS)-0.2% Tween was incubated for overnight at 4 0 C with a 1/500 anti-HA (12CA5; Boehringer Mannheim, Indianapolis, IN) in TBS-0.1% Tween with 3% bovine serum albumin followed by incubation with a 1/3,000 antimouse IgG, horseradish peroxidase-linked antibody (Amersham, Arlington Heights, IL) in TBS-0.1% Tween with milk. Reactive bands were detected using an ECL system (Amersham).

Rat-1 cell transfection and soft agar assay. Rat-1 cells plated at 1 x 106per 100-mm dish in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) were transfected with 3pmol of either pHeBo-CMV or pHeBo-CMV-MUM1-HA by modified calcium phosphate method (29).

Forty-eight hours after transfection, cells were reseeded into five 100-mm dishes and cultured in DMEM supplemented with 10% FBS containing 600lg/ml of G418. G418-resistant colonies were isolated after 10 days of selection. Analysis of clonogenecity was performed as described previously (29).

In brief, Rat-1 cells were plated in triplicate agar plates in DMEM plus 10% FBS) at 2.5 x 103, 5 x 103, and 1 x 104 cells per dish onto 35-mm agar plates containing DMEM, FBS, and 0.5% agar. After incubation for 2 weeks at 37 0 C, 5% CO,, colonies larger than 0.25mm were counted.

GenBank accession number. MUMlcDNA: U63738; 5' promoter: M- M- I M WO 97/45106 PCTfUS97/09065 -64- U63739.

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23. Kawano, M. et al. Autocrine generation and requirement WO 97/45106 PCT/US97/09065 -67of BSF-2/IL-6 for human multiple myelomas. Nature 332, 83-85 (1988).

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28. Chang, Ye, Chaganti, R.S.K. and Dalla- Favera, R. BCL-6, a POZ?zinc-finger protein, is a sequencespecific transcriptional repressor. Proc. Natl. Acad. Sci USA 93, 6947-6952 (1996).

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Opposite regulation of gene transcription and cell proliferation by c-Myc and Max. Proc. Natl. Acad. Sci USA 90, 2935-2939 (1993).

Y D WO 97/45106 PCT/US97/09065 -68- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Dalla-Favera, Riccardo (ii) TITLE OF INVENTION: IDENTIFICATION OF GENES ALTERED IN MULTIPLE MYELOMA (iii) NUMBER OF SEQUENCES: 22 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Cooper Dunham LLP STREET: 1185 Avenue of the Americas CITY: New York STATE: New York COUNTRY: U.S.A.

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REGISTRATION NUMBER: 28,678 REFERENCE/DOCKET NUMBER: 50995-A-PCT (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (212) 278-0400 TELEFAX: (212) 391-0525 WO 97/45106 PCT/US97/09065 -69- INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Leu Arg Gin Trp Leu Ile Asp Gin Ile Asp Ser Gly Lys Tyr Pro Gly Leu Val Trp Glu Asn Glu Glu Lys 25 Tyr Asn 40 Ser Ile Phe Arg Ile Pro Trp Ala Ala Leu Lys His Ala Gly Lys Gin Asp Arg Glu Glu Asp Phe Lys Lys Pro Ala Trp Ala Leu Phe 55 Lys Gly Lys Phe Arg Glu Gly Ile Asp Asp Pro Pro Thr 70 Trp Lys Thr Arg Leu 75 Glu Arg 90 Arg Cys Ala Leu Asn Lys Ser Asn Asp Phe Glu Glu Leu Val Ser Asp Pro Tyr Lys Val Tyr Arg Ile 100 105 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid STRANDEDNESS: single Ser Gin Leu Asp Ile Val Pro Glu WO 97/45106 WO 9745106PCT/US97/09065 TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Lys Leu Arg Gin Trp Leu Ile Asp Gin Ile Asp Ser Gly Lys Tyr Pro 1 Gly Leu Val Trp Lys His Ala Gly Phe Lys Ala Trp 5 Glu 10 Ser Asn Glu Glu Lys 25 Val Phe Arg Lys Gin Asp Ala Leu Phe 55 Pro Thr Trp Tyr Asn Arg Glu Giu 40 Lys Asp Ile Pro Trp Ala Ala Leu Gly Ile Asp Ala Leu Asn Leu Asp Ile Gly Lys Phe Pro Arg Glu Arg Cys Lys Lys Asp Pro Lys Thr Arg 70 Giu Leu 75 Ser Asn Asp Giu Leu Val Glu Arg Ser Tyr Arg Ile Val Pro Giu 105 Gin Ser Asp Pro Tyr Lys Val 100 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 97/45106 WO 9745106PCT/US97/09065 -71- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Arg Met Arg Pro Trp Leu Giu Met Gin Ile Asn Ser Asn Gin Ile Pro

I

Gly 10 Met Leu Ile Trp Ile Asn Lys Giu Giu 25 Asp Ile Phe Gin is Ile Pro Trp Aia Cys Leu Giy Giu Lys Lys His Aia Phe Arg Ser Giu Pro Asp Ala LYS His Gly Trp 40 Thr Ile Asn Lys Asp Ala Trp Ala Ile His 55 Gly Arg Tyr Ser Pro Lys Thr Trp 70 Asp Ile Glu Giu Lys Ala Asn Phe 75 Lys Arg Cys Ala Met Asn Giy Leu Pro Vai Lys Asp Gin Ser Arg Met Leu Pro Pro Arg Asn Lys Ser Ser Ala Val Arg Val Tyr 100 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Arg Met Arg Pro Trp Leu Glu Glu Gin Ile Asn Ser Asn Thr Ile Pro 1 5 10 Gly Leu Lys Trp Leu Asn Lys Glu Lys Lys Il~e Phe Gin Ile Pro Trp 25 WO 97/45106 WO 9745106PCT[US97/09065 -72 Trp 40 Met His Ala Ala Arg His Gly Asp Val Giu Lys Asp Ala Pro Leu Phe Arg 50 Asn Trp Ala Ile Thr Gly Lys His Gin Pro Gly Val. Asp Lys Pro Asp Pro Lys Thr 70 Trp Lys Ala Asn Phe 75 Arg Cys Ala Met Asn Ser Leu Pro Asp Giu Giu Vai Lys Asp Lys Ser Ile Lys Lys Gly Asn Asn Ala Phe Arg Vai Tyr ArgI 100 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: i07 amino acids TYPE: amino acid STRAN~DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide Leu Pro Leu (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Arg Gin Trp Leu Ile Giu Gin Ile Asp Ser Ser Met Tyr Pro is Gly Leu Ile Trp, Giu Asn Giu Glu Lys Ser Met Phe Arg Ile Pro Trp Lys His Ala Gly Lys Gin Asp Tyr Asn Gin Giu 40 Phe Lys Ala Trp Aia Val Phe Lys Gly Lys Phe Val. Asp Ala Ser Ile Lys Giu Gly Asp Lys WO 97/45106 PCT/US97/09065 -73- Ala Glu Pro Ala Thr Trp Lys Thr Arg Leu Arg Cys 70 75 Ala Leu Asn Lys Ser Pro Asp Phe Glu Glu Val Thr Asp Arg 90 Ser Gin Leu Asp Ile Ser Glu Pro Tyr Lys Val Tyr Arg Ile Val Pro Glu 100 105 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Lys Leu Arg Asn Trp Val Val Glu Gin Val Glu Ser Gly Gin Phe Pro Gly Val Cys Trp Asp Asp Thr Ala Lys 25 Thr Met Phe Arg Ile Pro Trp Ala Ala Phe Lys His Ala Gly Lys Gin Asp Phe Arg 40 Glu Asp Gin Asp Phe Lys Ala Trp Ala Ile Phe 55 Lys Gly Lys Tyr Lys Glu Gly Asp Thr Gly Gly Pro Ala Val Ser Ser Glu Phe Lys Trp Lys Thr Arg 70 Leu Arg Cys Ala Leu Asn Glu Val Pro Glu Arg Gly Arg Met Asp Val Ala WO 97/45106 PCT/US97/09065 -74- Glu Pro Tyr Lys Val Tyr Gin Leu Leu Pro Pro 100 105 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 106 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) Arg 1 Gly Lys SEQUENCE DESCRIPTION: SEQ ID NO:7: Ile Leu Pro Trp Leu Val Ser Gin Leu 5 10 Val Ala Trp Val Asn Lys Ser Arg Thr 25 His Gly Leu Arg Gin Asp Ala Gin Gin Asp Leu Gly Gin Leu Glu Arg Phe Arg Glu Asp Phe Val Pro Gly Gin Ala Trp Ala Glu Ala 40 Thr Gly 55 Lys Arg Ala Tyr Pro Asp Asn Phe Arg 75 Ser Ala Ile Pro Trp Gly Ile Phe Arg Asp Lys Leu Asn Arg Pro His Asp Leu Pro Thr Lys Glu Gly Leu Arg Tyr Leu Ala Glu Asp Arg Ser Lys Asp 90 Glu Phe Val Asn Ser Pro His Lys Ile 100 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: I I WO 97/45106 PCT/US97/09065 LENGTH: 95 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Lys Arg Leu Cys Gin Ser Thr Ile Tyr Trp Asp Gly Pro Leu Ala Leu Cys Asn Asp Arg Phe Asp Thr Gin Pro Asn Lys Leu Arg Asp Gin Thr Cys Lys Leu Ala His His Gin Phe Leu Ser Glu Leu Gin Ala Gly Arg Phe Pro Ser Leu Pro Arg Asp Pro Gin Arg 70 Phe Gin Val Thr Leu Phe Gly Glu Glu Gin Arg Lys Leu Ile Thr Ala His Val Pro Leu Leu Ala Arg Gin Leu Tyr Tyr Phe Ala Gin Gin Asn Ser INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 95 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: WO 97/45106 WO 9745106PCT/US97/09065 -76- Lys Arg Leu Cys Gin Ser Arg Ile Tyr Trp Asp Gly Pro Leu Ala Leu Cys Ser Asp Arg Pro Asn Lys Leu Glu 25 Ser Glu 40 Arg Asp Gin Thr Cys Lys Leu Phe Asp Thr Gin Gin Phe Leu Leu Gin Val Phe Ala His His Gly Arg Pro Ala Pro Arg Phe 55 Gin Val Thr Leu Cys Phe Gly Giu Glu Phe Pro Asp Pro Gin Arg Gin 70 Arg Lys Leu Ile 75 Thr Ala His Val Glu Pro Leu Leu Ala Arg Gin Leu Tyr Tyr INFORMATION FOR SEQ ID NO:l0: Ala Gin Gin Asn Thr SEQUENCE CHARACTERISTICS: LENGTH: 96 amino acids TYPE: amino acid STRAflDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:iO: Lys Arg Leu Cys Gin Gly Arg Val Phe Cys Ser Gly Asn Ala Val Val 1 5 10 Cys Lys Gly Arg Pro Asn Lys Leu Glu Arg Asp Glu Vai Val Gin Val Phe Asp Thr Ser Gin Phe Phe Arg Glu Leu Gin Gin Phe Tyr Asn Ser WO 97145106 WO 9745106PCTIUS97/09065 -77- Gin Gly Arg Leu Pro Asp Gly Arg 55 Val Val Leu Cys Phe Gly Giu Glu Phe Pro Asp Met Ala Pro Leu Arg Ser Lys Leu 70 75 Glu Gin Leu Tyr Val Arg Gin Leu Ala Giu Glu 90 Ile Leu Vai Gin Ile Ala Giy Lys Ser Cys INFORMATION FOR SEQ ID NO:ii: SEQUENCE CHARACTERISTICS: LENGTH: 96 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ii: Gin 1 Arg Leu Cys Pro Ile Pro Ile Ser Trp Asn Ala Pro Gin Ala Pro Pro Gly Pro Giy 20 Pro His Leu Leu Pro Ser 25 Asn Giu Cys Val Glu Leu Phe Gin Gly Phe Arg Thr Ala Tyr Phe Cys Arg 40 Asp Leu Val Arg Tyr Leu Gly Pro Pro Pro Lys Phe Gin Val Thr Leu Phe Trp Glu Glu Ser His Giy Ser Ser His 70 Thr Pro Gin Asn Leu 75 Ile Thr Val Lys Met Giu Gin Ala Phe Ala Arg Tyr Leu Leu Giu Gin 90 Thr Pro Giu Gin Gin WO 97/45106 -78- INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 100 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: PCT/US97/09065 Gin 1 Arg Leu Gly His Cys His Thr 5 Tyr Trp 10 Asp Gly 25 Ala Val Ser Glu Glu Leu Leu Pro Asn Ser Glu Gly Gly Val Gly His Gly Pro Phe Asp Leu Gly 40 Glu Val Pro Lys Asp Lys Leu Ile Thr Pro Phe Ile Val Asp Phe Thr Glu Gly Ser Gly Arg Ser Pro Arg Tyr Leu Trp Phe Cys Val Gly Glu Ser Trp Met Val Lys Val Val Pro Gin Asp Gin Pro Thr Lys Arg Leu Pro Thr Cys Leu Arg Ala Leu Val Glu Met Ala Arg Val Gly Gly 100 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 5176 base pairs TYPE: nucleic acid WO 97/45106 PCTIUS97/09065 -79- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: NAME/KEY: CDS LOCATION: 217..1569 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCCTGACCAA CATGGTAAAA CCCCATCTCT GCTAAAACTA CAAAAAATTA GCTGGATGTG GTGGCAGGGA ACCTGTCATC CCAGCTAGTT GGGAGACTGA GGCAGGAGAA TCGCTCGATC TTGGGACCCA CCGCTGCCCT CAGCTCCGAG TCCAGGGCGA GTGCAGAGCA CAGCGGGCGG AGGACCCCGG GCGCGGGCGC GGACGGCACG CGGGGC ATG AAC CTG GAG GGC GGC Met Asn Leu Glu Gly Gly GGC CGA GGC GGA GAG TTC GGC ATG AGC GCG GTG AGC TGC GOC AAC GGG Gly Arg Gly Gly Glu Phe Gly Met Ser Ala Val Ser Cys Gly Asn Gly 15 AAG CTC CGC CAG TGG CTG ATC GAC CAG ATC GAC AGC GGC AAG TAC CCC Lys Leu Arg Gln Trp Leu Ile Asp Gin Ile Asp Ser Gly Lys Tyr Pro 30 GGG CTG GTG TGG GAG AAC GAG GAG AAG AGC ATC TTC CGC ATC CCC TGG Gly Leu Val Trp Glu Asn Glu Glu Lys Ser Ile Phe Arg Ile Pro Trp 40 45 AAG CAC GCG GGC AAG CAG GAC TAC AAC CGC GAG GAG GAC GCC GCG CTC Lys His Ala Gly Lys Gin Asp Tyr Asn Arg Glu Glu Asp Ala Ala Leu 60 65 TTC AAG GCT TGG GCA CTG TTT AAA GGA AAG TTC CGA GAA GGC ATC GAC Phe Lys Ala Trp Ala Leu Phe Lys Gly Lys Phe Arg Glu Gly Ile Asp 282

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WO 97/45106 PCT/US97/09065 AAG CCG GAC CCT CCC ACC TGG AAG ACG CGC CTG CGG TGC GCT TTG AAC Lys Pro Asp Pro Pro Thr Trp Lys Thr Arg Leu Arg Cys Ala Leu Asn 95 100 AAG AGC AAT GAC TTT GAG GAA CTG GTT GAG CGG AGC CAG CTG GAC ATC Lys Ser Asn Asp Phe Glu Glu Leu Val Glu Arg Ser Gin Leu Asp Ile 105 110 115 TCA GAC CCG TAC AAA GTG TAC AGG ATT GTT CCT GAG GGA GCC AAA AAA Ser Asp Pro Tyr Lys Val Tyr Arg Ile Val Pro Glu Gly Ala Lys Lys 120 125 130

GGA

Gly 135 GCC AAG Ala Lys CAG CTC ACC CTG GAG GAC CCG CAG ATG TCC ATG AGC CAC Gin Leu Thr Leu Glu Asp Pro Gin Met Ser Met Ser His 140 145 150 ATG ACA ACG CCT TAC CCT TCG CTC CCA GCC CAG CAG GTT Met Thr Thr Pro Tyr Pro Ser Leu Pro Ala Gin Gin Val 155 160 165 CCC TAC ACC Pro Tyr Thr 666 714 762 CAC AAC TAC ATG ATG CCA CCC CTC GAC CGA AGC TGG AGG GAC TAC GTC His Asn Tyr Met Met Pro Pro Leu Asp Arg Ser Trp Arg Asp Tyr Val 170 175 180 CCG GAT Pro Asp CAG CCA CAC CCG GAA ATC CCG TAC CAA TGT CCC ATG ACG TTT Gin Pro His Pro Glu Ile Pro Tyr Gin Cys Pro Met Thr Phe 185 190 195 CGC GGC CAC CAC TGG CAA GGC CCA GCT TGT GAA AAT GGT TGC Arg Gly His His Trp Gin Gly Pro Ala Cys Glu Asn Gly Cys 205 210 GGA CCC Gly Pro 200 810 858 906 CAG GTG ACA GGA ACC TTT TAT GCT TGT GCC CCA CCT GAG TCC CAG GCT Gin Val Thr Gly Thr Phe Tyr Ala Cys Ala Pro Pro Glu Ser Gin Ala 215 220 225 230 CCC GGA GTC CCC ACA GAG CCA AGC ATA AGG TCT GCC GAA GCC TTG GCG Pro Gly Val Pro Thr Glu Pro Ser Ile Arg Ser Ala Glu Ala Leu Ala WO 97/45106 PCTIUS97/09065 -81- 235 240 245 TTC TCA GAC TGC CGG CTG CAC ATC TGC CTG TAC TAC CGG GAA ATC CTC 1002 Phe Ser Asp Cys Arg Leu His Ile Cys Leu Tyr Tyr Arg Glu Ile Leu 250 255 260 GTG AAG GAG CTG ACC ACG TCC AGC CCC GAG GGC TGC CGG ATC TCC CAT 1050 Val Lys Glu Leu Thr Thr Ser Ser Pro Glu Gly Cys Arg Ile Ser His 265 270 275 GGA CAT ACG TAT GAC GCC AGC AAC CTG GAC CAG GTC CTG TTC CCC TAC 1098 Gly His Thr Tyr Asp Ala Ser Asn Leu Asp Gin Val Leu Phe Pro Tyr 280 285 290 CCA GAG GAC AAT GGC CAC AGG AAA AAC ATT GAG AAC CTG CTG AGC CAC 1146 Pro Glu Asp Asn Gly His Arg Lys Asn Ile Glu Asn Leu Leu Ser His 295 300 305 310 CTG GAG AGG GGC GTG GTC CTC TGG ATG GCC CCC GAC GGG CTC TAT GCG 1194 Leu Glu Arg Gly Val Val Leu Trp Met Ala Pro Asp Gly Leu Tyr Ala 315 320 325 AAA AGA CTG TGC CAG AGC ACG ATC TAC TGG GAC GGG CCC CTG GCG CTG 1242 Lys Arg Leu Cys Gin Ser Thr Ile Tyr Trp Asp Gly Pro Leu Ala Leu 330 335 340 TGC AAC GAC CGG CCC AAC AAA CTG GAG AGA GAC CAG ACC TGC AAG CTC 1290 Cys Asn Asp Arg Pro Asn Lys Leu Glu Arg Asp Gin Thr Cys Lys Leu 345 350 355 TTT GAC ACA CAG CAG TTC TTG TCA GAG CTG CAA GCG TTT GCT CAC CAC 1338 Phe Asp Thr Gin Gin Phe Leu Ser Glu Leu Gin Ala Phe Ala His His 360 365 370 GGC CGC TCC CTG CCA AGA TTC CAG GTG ACT CTA TGC TTT GGA GAG GAG 1386 Gly Arg Ser Leu Pro Arg Phe Gin Val Thr Leu Cys Phe Gly Glu Glu 375 380 385 390 TTT CCA GAC CCT CAG AGG CAA AGA AAG CTC ATC ACA GCT CAC GTA GAA 1434 Phe Pro Asp Pro Gin Arg Gin Arg Lys Leu Ile Thr Ala His Val Glu 395 400 405 WO 97/45106 WO 9745106PCTIUS97/09065 -82- CCT CTG CTA GCC AGA CAA CTA TAT TAT TTT GCT CAA CAA AAC AGT GGA Pro Leu Leu Ala Arg Gin Leu Tyr Tyr Phe Ala Gin Gin Asn Ser Giy 410 415 420 CAT TTC CTG AGG GGC TAC GAT TTA CCA GAA CAC ATC AGC AAT CCA GAA His Phe Leu Arg Giy Tyr Asp Leu Pro Giu His Ile Ser Asn Pro Glu 425 430 435 GAT TAC CAC AGA TCT ATC CGC CAT TCC TCT ATT CAA GAA TGAAAAATGT Asp Tyr His Arg Ser Ile Arg His Ser Ser Ile Gin Giu 440 445 450 14 82 1530 1579 CAAGATGAGT GGTTTTCTTT TTCCTTTTTT GTCTTGCTCT GTCTCCCAGG CTGGAGTGCA GCCTCCTGGG TTCAAGAGAC TCTCCTGCCT TGAGCCACTG CACCCACCCA AGACAAGTGA AGCGTCCAAT TGACTGCCCT CTTACTGTTT AGCGGTTGAG GAGAATTGCG GCGAGACAAG ATGAGCTTAT TTCAAAAGGA AGGGTGGCTT CATTGATGAT CACTGTGAAA ATTGACCAAG TTTAATTTGT TGTAGATTAG GTCTTGCTGG ACACTGACTA GAGTGATGAC TGCTTGTAGG ATGTAAATTG AAGAAGCCTC ACACGTAAAA TTCTTGTGGA AGACACTTGC TGAGTGAAGG AGCCTTGGGG AGGCCCATCC CCCACCTGCC CACCCTCCTT CCCATTGGCT TTCTCTCCTT CCTATTTTCT TGAGTCAAAA AACATGAGCG

TTTTTTTTTT

GTGACACAAT

CAGCCTCCCT

TTTTCATTGT

TGAGGAACTC

CATGGAAAAT

TGCATTTTCT

TGATGTGTTT

AAGACAGAGA

TATGTCTGTG

GAAATGTATT

AAATGAATCT

AGCGGTTTCC

GGCCTTTCCT

CTACTCTTGG

TTTTGATACG

CTCAGCTCAC

GGTAGCTGGG

AAATATTTGA

AGAAGTGGAG

CAGTGACATC

TGTGTTCTGT

ACATTTACTG

AAACTTGCCT

CCATTTCTCA

AATGTATGTA

TTGACTGAAG

GAGATACGGG

TGTGACCTCC

ATTACAGGTG

CTTTAGTGAA

ATTTCAGTTC

TGATTGGCAG

AGACTGCCAT

AAATGCGCTC

TTCAGTATTG

GGGAAGTAAG

GGAGCTGCAG

CCGTGCCTGT

1639 1699 1759 1819 1879 1939 1999 2059 2119 2179 2239 2299 2359 2419 2479 TGGTGTGGGT CCCTCTGCCC

GGAAGCCAGT

ATGGGACATT

TAGTAAACTT

TTTGTCTGTC

WO 97/45106 WO 9745106PCT/US97/09065 -83- CTACAATCTA GTAATGTCTA AGTAATGGTT AAGTTTTCTT GTTTCTGCAT CTCATTCTTT AGAGATGCTA TCTTAATACT TGAACTGTTG GTGCCACGCT CCTCTGTTTG GAGGAGCCGG GGACTCCCAG GGAGCTGACT ACGGAACTGC GCTTCCTCGT GCCAATTATA CTTCTATTTA TAGAAATCCC TTTGTGGTTT TGAGAAAGTA ACTACAGGAT ATTTACTATT GTGTGTTCCT GCTTTTCTAA CTGCTGTATC CCTACATTAC GAAACATTAT TTTTTAATGT TTAATATTAA TATTAACTAC GATCTCCTTT TTTCCCAGCC TATTCTTCCA ACACATTTCA TGGAGTCATC TTGCACACAC ACCTCGTTCT GCTCAGAGGC GACATTTCAT TAGCCATGCA

%AATTCTTCG

CCCTTCTGTC

TTTGGCTGTC

GCTGGAGAGC

ACTCAGTG

GTTTGACAGG

AAAGACCTCC

CAGCAGTAGA

ACTCCCAGGA

TGGATATTTT

ACAGTTCAGC

TTAAAAAGTT

AGAAAAGACA

CAAATTCTCC

CTTTTCTGTA

TTTTCATGCA

CTTGCTGTGG

ACATGGATAT

CATAAAGAAG

CAAGTACTTA

CAGCGATCAG

PCTGCCAGGA

GGCTGTTTCT

GCCTTAAAAT

ACTTGCTTAA

CTGGGGCGTC

TTCAGCAGAA

AAATTCATTC

CTTTATCAAG

TCTAATATTA

AACAGTAGAG

TCTCTAAAAG

AATATACATA

GTGCTCTTTG

AGCTCCACTG

GTATTGGGCA

AGTATTTTGG

%AGAAATTAA

%CTATCTGTT

CCATGGCGAC

CCCACCACTG

GCTTATTTCA

TACTTGGCTT

GTATACCTAT

ACCTCCAGGC

GATTGCGTTA

AACAAGCACC

CTTAGTGAGC

AAGTCAGAAT

AACAGCAAAA

TGTCCACAAG

AACTTAAAAA

TAGCTAAACA

CCATGTACCC

CTTTTTGACC

GGAACATAAA

CCCTTCCTCT

ACTAAAGGAG

GAAGCAGGAT

TCTGTTCTAT

TTTCCAA-ATG

CACTTACATT

CGTTTCTCAT

GCTCTCAAAT

TAGTAAGTGC

AGTGAGCACT

ATTAATACAA

AAATAAAAAG

AAGGGGTGTT

GAAAACCTCA

GTGAAGATTT

AGTAGGGTTT

2539 2599 2659 2719 2779 2839 2899 2959 3019 3079 31.39 3199 3259 3319 3379 3439 3499 3559 3619 3679 GCAGACTGTG TTTCGTGAAC GGTATATTAT CCTAAGGGAA TGCAGTGATG TATACATCTT ATAGATGCAA GATAAAGATG ATATTAAGAA CTGCTGTTTC ACGGGGCCCT TACCTGTGAC CCTCTTTGCT 33 3739 WO 97/45106 WO 9745106PCTIUS97/09065 GAAGAATATT TAACCCCACA GCACCCTGTC TTCTTAATTC CTTCTTTGTT TTTTTAAATA GCTTCTTGGC TGGGGCCACT TTTAAAATTC TGAGTGATCC CAGGGTTAGG AATCCTAGCA TTGTCTTGAT AAAGTGGAAT GTTGAAGATT TGAGGACTTG CCCCAGGTGC ATTTTCTTGG CTCAAGCAGT AATTAATATC CAACTGAAAC CTAGGAAGCC

CAGCACTTCA

TCCAAGCGGA

TTATGCTGCT

ACCTCCTTTC

AGGGTATGAC

CTTGTCTCAG

TGGCAAACTA

TTAAAGAGCA

TTTATGTCTT

TCCTGGAACA

CCTGAGTCCT

-84-

AAGAAGCTGT

TGCTCCATTT

TTAACAGTGG

CTATCTTTAC

CTAGGGAATG

GACTCTGAAA

GAATTTAGTT

CTGGGTCATA

GTTCTTGAGA

CTTGGAAGTC

CAATTGCTTT

AGCTGAATTT

ATCTATGTGT

AACTAGCTAT

AGGAACGGCT

TGTACTCAGT

TGGAAAAAAT

TTTTGTATAT

TGTCTCAGGA

GTGACTTCTT

TCTGGAAAAT

ATGTTGACTT

GGAAATAACT

TCCTCATTCC

GGACAGTGCT

GTATGTGTCT

TTAGGAAAAC

3799 3859 3919 3979 4039 4099 4159 4219 4279 4339 4399 CTATAGAGAA CCAAGTGACC GACTCATTTA GAGCGAAAAC AGGAGAGTTA GTCGCCCTAC AGAAAACCCA GCTAGACTAT TGGGTATGAA CTAAAAAGAG ACTGTGCCAT GGTGAGAAAA 4459 ATGTAAAATC CTACAGTGGA ATGAGCAGCC CTTACAGTGT TGTTACCACC AAGGGCAGGT 4519 AGGTATTAGT GTTTGAAAAA GCTGGTCTTT GAGCGAGGGC ATAAATACAG CTAGCCCCAG 4579 GGGTGGAACA ACTGTGGGAG TCTTGGGTAC TCGCACCTCT TGGCTTTGTT GATGCTCCGC 4639 CAGGAAGGCC ACTTGTGTGT GCGTGTCAGT TACTTTTTTA GTAACAATTC AGATCCAGTG 4699 TAAACTTCCG TTCATTGCTC TCCAGTCACA TGCCCCCACT TCCCCACAGG TGAAAGTTTT 4759 TCTGAAGTGT TGGGATTGGT TAAGGTCTTT ATTTGTATTA CGTATCTCCC CAAGTCCTCT 4819 GTGGCCAGCT GCATCTGTCT GAATGGTGCG TGAAGGCTCT CAGACCTTAC ACACCATTTT 4879 GTAAGTTATG TTTTACATGC CCCGTTTTTG AGACTGATCT CGATGCAGGT GGATCTCCTT 4939 GAGATCCTGA TAGCCTGTTA CAGGAATGAA GTAAAGGTCA GTTTTTTTTG TATTGATTTT 4999 WO 97/45106 WO 9745106PCTIUS97/09065 CACAGCTTTG AGGAACATGC ATAAGAAATG TAGCTGAAGT AGAGGGGACG TGAGAGAAGG GCCAGGCCGG CAGGCCAACC CTCCTCCAAT GGAAATTCCC GTGTTGCTTC AAACTGAGAC AGATGGGACT TAACAGGCAA TGGGGTCCAC TTCCCCCTCT TCAGCATCCC CCGTACC 5059 5119 5176 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 451 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Asn Leu GTh Gly Gly Gly Arg Gly Gly Giu Phe Gly Met Ser Cys Gly Asn Ser Gly Lys Tyr 10 Gin Ser Ala Gly Lys Leu Arg 25 Pro Gly Leu Val Trp Leu Ile Asp Gin Ile Giu Lys Ser Asp Trp GTh Asn Ile Phe Arg His Ile Pro Trp Glu Lys 55 Phe Ala Gly Lys Gin Asp Tyr Asn Arg Glu Phe Asp Ala Ala Leu 70 Arg Glu Gly Ilie Asp Lys Ala Leu Asn Lys Lys Ala Trp Pro Asp Pro 90 Ser Asn Asp Leu Thr Phe Lys Gly Lys Trp Lys Thr Arg Glu Leu Val Glu Leu Arg Cys Phe Giu Arg Ser Gin Leu Asp Ile Ser Asp Pro Tyr Lys Val Tyr Arg Ile Val 115 120 125 WO 97/45106 WO 9745106PCTIUS97/09065 -86- Pro Giu 130 Gly Ala Lys Lys Gly Ala Lys Gin Leu 135 Thr 140 Leu Giu Asp Pro Gin 145 Met Ser Met Ser His Pro Tyr Thr Met 150 Thr Pro Tyr Pro Ser 160 Leu Pro Ala Gin Gin 165 Val His Asn Tyr Met Pro Pro Leu Asp Arg 175 Ser Trp Arg Gin Cys Pro 195 Asp 180 Tyr Val Pro Asp Gin 185 Pro His Pro Giu Ile Pro Tyr 190 Gin Gly Pro Met Thr Phe Gly Arg Gly His His Trp 205 Ala Cys 21i0 Giu Asn Gly Cys Val Thr Gly Thr Tyr Ala Cys Ala Pro Giu Ser Gin Pro Gly Vai Pro Giu Pro Ser Ile Arg 240 Ser Ala Giu Ala Ala Phe Ser Asp Arg Leu His Ile Cys Leu 255 Tyr Tyr Arg Giy Cys Arg 275 Ile Leu Val Lys Giu 265 Leu Thr Thr Ser Ser Pro Giu 270 Asn Leu Asp Ile Ser His Gly His 280 Thr Tyr Asp Ala Ser 285 Gin Val 290 Leu Phe Pro Tyr Pro 295 Giu Asp Asn Gly His 300 Arg Lys Asn Ilie Giu 305 Asn Leu Leu Ser His 310 Leu Giu Arg Giy Vai 315 Val Leu Trp Met Pro Asp Gly Leu Tyr Ala 325 Lys Arg Leu Cys Gin Ser Thr 330 Ile Tyr Trp 335 Leu Giu Arg 350 Asp Gly Pro Leu Ala Leu Cys Asn Asp Arg Pro Asn Lys 340 345 WO 97/45106 WO 9745106PCTfUS97/09065 -87- Thr Gin Gin Phe Asp Gin Thr 355 Gin Ala Phe Cys Lys Leu Phe Asp 360 Arg Leu 365 Phe Ser Giu Leu Gin Val Thr Ala His His 370 Gly 375 Phe Ser Leu Pro Arg 380 Arg Leu 385 Ile Cys Phe Gly Glu Giu 390 Giu Pro Asp Pro Gin Arg Lys Leu 400 Thr Ala His Val 405 Ser Pro Leu Leu Ala 410 Arg Arg Gin Leu Tyr Tyr Phe 4215 Ala Gin Gin Asn 420 Asn Gly His Phe Leu 425 His Gly Tyr Asp Leu Pro GZlu 430 His Ser Ser His Ilie Ser 435 Pro Giu Asp Tyr 440 Arg Ser Ile Ile Gin Glu 450 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 154 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID TTTTCTCTCT ACAGTCACCT CCCTGTTTAC CAAAGATAAT CACAATAAGT CCAGTTTACT TACAAAACAA GTTTAGTTAT TAGAGGAAAC TAAAACTTCA GGATTCAGTC CAGATAATTT TTAAAAACTC TAAAACAATG GACAGGGCTA GAAT INFORMATION FOR SEQ ID NO:i6:

~I

A WO 97/45106 PCT/US97/09065 -88- SEQUENCE CHARACTERISTICS: LENGTH: 75 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TGGGCTCGGC CTGGTGGGGC AGCCACAGCG GGACGCAGTA GTGAAAGTCC AGTTTACTTA CAAAACAAGT TTAGT INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 122 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TGGGCTCGGC CTTGGTGGGG CAGCCACAGC GGGACGCAAG TAGTGAGGGC ACTCAGAACG CCACTCAGCC CCGACAGGCA GGGCACGAGG AGGCAGCTCC TCACCCTCCC TTTCTCTTTT WO 97/45106 PCT/US97/09065 -89- INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 77 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TATTAGAGGA AACTAAAACT TCAGGATTCA GCAGGGCATG AGGAGGCAGC TCCTCACCCT CCCTTTCTCT TTTGTAC 77 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TACTCGCACC TCTTGGCT 18 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs WO 97/45106 PCT/US97/09065 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID CTGGAGAGCA ATGAACGG 18 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: AGGTACTTTC AGTTTCATAT T 21 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 97/45 106 PCT1US97/09065 -91- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Met Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Gly Pro Gly 1 5 10 S Pro

Claims

2. The method of claim 1, wherein step is performed by Southern blotting.
3. The method of claim 1, wherein step is performed by WO 97/45106 PCTtUS97/09065 -93- polymerase chain reaction with appropriate probes.
4. The method of claim 1, wherein the genomic library is a phage vector library. The method of claim 4, wherein the genomic DNA fragments are generated by cleaving genomic DNA from cells of the subject with an appropriate restriction enzyme.
6. The method of claim 5, wherein the restriction enzyme is BamHI.
7. The method of claim 5, wherein the restriction enzyme is Sau3AI.
8. The method of claim 1, wherein the probe of step is a human IgH J region JH probe.
9. The method of claim 1, wherein the probe of step is a human IgH CA probe. The method of claim 1, wherein the probe of step (d) is a human IgH Cy2 probe.
11. The method of claim 1, wherein the chromosomal breakpoint identified is a t(6;14)(p25;q32) translocation.
12. The method of claim 1, wherein the chromosomal breakpoint identified is a t(l;14) translocation.
13. A method to identify a gene other than the immunoglobulin gene which is located in chromosome 14, altered by a chromosomal breakpoint detected in WO 97/45106 PCT/US97/09065 -94- a subject suffering from multiple myeloma which comprises steps of: a) selecting a probe having a sequence of a chromosome other than chromosome 14, identified at the chromosomal breakpoint detected in a subject suffering from multiple myeloma, wherein said probe is capable of hybridizing to the unique sequence of the gene other than the immunoglobulin gene altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma; b) contacting said probe with mRNA isolated from a cell under conditions permitting formation of a complex between said probe and the mRNA; c) isolating the complex resulting from step d) determining the sequence of the mRNA in the isolated complex, thereby determining the identity of the gene.
14. The method of claim 13, wherein step comprises steps of: i) synthesizing complementary DNA to the mRNA; and ii) performing sequence analysis of the complementary DNA to determine the sequence of the mRNA. A gene identified by the method of claim 13.
16. The gene of claim 15 designated MUM-I.
17. The gene of claim 15 designated MUM-2.
18. The method of claim 13, wherein the gene identified comprises a nucleic acid encoding a MUM protein. WO 97/45106 PCT/US97/09065
19. The method of claim 18, wherein the MUM protein is MUM-1. The method of claim 18, wherein the MUM protein is MUM-2.
21. An isolated nucleic acid molecule encoding a MUM protein.
22. An isolated nucleic acid molecule of claim 21, wherein the nucleic acid molecule is a DNA molecule.
23. The isolated DNA molecule of claim 22, wherein the DNA molecule is a cDNA molecule.
24. The isolated DNA molecule of claim 21, wherein the DNA molecule is a cDNA molecule having the nucleotide sequence shown in Figure 5B (SEQ. ID NO 13). The isolated DNA molecule of claim 22, wherein the DNA molecule is genomic DNA molecule.
26. The isolated nucleic acid molecule of claim 21, wherein the nucleic acid molecule is an RNA molecule.
27. An isolated nucleic acid molecule of claim 21, wherein the nucleic acid molecule encodes a human MUM-1 protein.
28. An isolated nucleic acid molecule of claim 21, wherein the nucleic acid molecule encodes a human MUM-2 protein. WO 97/45106 PCT/US97/09065
29. -96- An isolated nucleic molecule of claim 27, wherein the human MUM-1 protein has substantially the same amino acid sequence as shown in Figure 5B (SEQ. ID NO 14). An isolated nucleic molecule of claim 27, wherein the human MUM-1 protein has the amino acid sequence as shown in Figure 5B (SEQ. ID NO 14). An isolated nucleic acid molecule of claim 21 operatively linked to a promoter of RNA transcription.
31.
32.
33.
34. A vector comprising the nucleic claim 21. A vector comprising the nucleic claim 23. A vector comprising the nucleic claim A vector of claim 33, wherein plasmid. acid molecule of acid molecule of acid molecule of the vector is a
36.
37. The plasmid of claim 35, designated pcMUM1-1.6a (ATCC Accession No. 97579). The plasmid of claim 35, designated pMUM1-2.4B/N (ATCC Accession No. 97578). The plasmid of claim 35, designated pMUM1-7.7B (ATCC Accession No. 97577). The plasmid of claim 35, designated pcMUM2-8 (ATCC
38.
39. WO 97/45106 PCT/US97/09065 -97-
41.
43. Accession No. 97580) A host cell comprising the vector of claims 32. The host cell of claim 40, wherein the cell is selected from a group consisting of a bacterial cell, a plant cell, and insect cell and a mammalian cell. A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a MUM protein. A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM protein. The nucleic acid probe of either of claims 42 or 43, wherein the MUM protein is MUM-1. The nucleic acid probe of either of claim 42 or 43, wherein the MUM protein is MUM-2. A DNA probe of claim 44 or A RNA probe of claim 44 or A genomic DNA probe of claim 44 or A nucleic acid probe of claim 44 or 45 labeled with a detectable marker.
44.
46.
47.
48.
49. WO 97/45106 PCT/US97/09065 -98- The nucleic acid probe of claim 49, wherein the detectable marker is selected from the group consisting of a radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.
51. A nucleic acid probe of claim 44, wherein the sequence of a nucleic acid molecule encoding a MUM-l protein is linked to a nucleic acid sequence capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule of human chromosome 14.
52. A nucleic acid probe of claim 45, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked to a nucleic acid sequence capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule of human chromosome 14.
53. A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule of claim 21 which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.
54. The nucleic acid probe of claim 53, wherein the isolated nucleic acid molecule encodes MUM-1. The nucleic acid probe of claim 53, wherein the isolated nucleic acid molecule encodes MUM-2.
56. The nucleic acid probe of claim 54, wherein the sequence of a nucleic acid molecule encoding a MUM-I WO 97/45106 PCT/US97/09065 -99- protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14.
57. The nucleic acid probe of claim 55, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14.
58. The nucleic acid probe of claim 56, wherein the specific break point comprises a portion of the t(6;14)(p25;q32) translocation.
59. The nucleic acid probe of claim 57, wherein the specific break point comprises a portion of a t(1;14) translocation. The nucleic acid probe of either of claims 58 or 59 labeled with a detectable marker.
61. The nucleic acid probe of claim 60, wherein the detectable marker is selected from the group consisting of a radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.
62. A method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-1 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-1 protein.
63. A method for detecting a predisposition to multiple WO 97/45106 PCT/US97/09065 -100- myeloma associated with the expression of a human MUM-2 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein.
64. The method of claim 62, wherein the rearrangement of nucleic acid encoding MUM-l protein is detected by contacting the nucleic acid from the sample with a MUM-I probe under conditions permitting the MUM-i probe to hybridize with the nucleic acid encoding MUM-i protein from the sample, thereby detecting the rearrangement of nucleic acid encoding MUM-I protein in the sample. The method of claim 63, wherein the rearrangement of nucleic acid encoding MUM-2 protein is detected by contacting the nucleic acid from the sample with a MUM-2 probe under conditions permitting the MUM-2 probe to hybridize with the nucleic acid encoding MUM-2 protein from the sample, thereby detecting the rearrangement of nucleic acid encoding MUM-2 protein in the sample.
66. The method of claim 64, wherein the MUM-i probe comprises a nucleic acid molecule of at least nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-I protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.
67. The method of claim 65, wherein the MUM-2 probe comprises a nucleic acid molecule of at least nucleotides which is complementary to a sequence of WO 97/45106 PCTIUS97/09065 -101- the isolated nucleic acid molecule encoding MUM-2 protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 1.
68. The method of claim 66, wherein the nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-l protein is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.
69. The method of claim 67, wherein the nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-2 protein is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 1. The method of claim 68, wherein the specific break point comprises a portion of the t(6;14) (p25;q32) translocation.
71. The method of claim 69, wherein the specific break point comprises a portion of a t(1;14) translocation.
72. The method of claim 62, which comprises: a. obtaining DNA from the sample of the subject suffering from multiple myeloma; b. performing a restriction digest of the DNA with a panel of restriction enzymes; WO 97/45106 PCT/US97/09065 -102- c. separating the resulting DNA fragments by size fractionation; d. contacting the resulting DNA fragments with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-l protein, wherein the sequence of a nucleic acid molecule encoding a MUM-l protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 and labeled with a detectable marker; e. detecting labeled bands which have hybridized to the nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-l protein, wherein the sequence of a nucleic acid molecule encoding a MUM-l protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 to create a unique band pattern specific to the DNA of subjects suffering from multiple myeloma; f. preparing DNA obtained from a sample of a subject for diagnosis by steps and g. comparing the detected band pattern specific to the DNA obtained from a sample of subjects suffering from multiple myeloma from step (e) and the DNA obtained from a sample of the subject for diagnosis from step to determine whether the patterns are the same or WO 97/45106 PCT/US97/09065 -103- different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.
73. The method of claim 63, which comprises: a. obtaining DNA from the sample of the subject suffering from multiple myeloma; b. performing a restriction digest of the DNA with a panel of restriction enzymes; c. separating the resulting DNA fragments by size fractionation; d. contacting the resulting DNA fragments with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-2 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 and labeled with a detectable marker; e. detecting labeled bands which have hybridized to the nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-2 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 to create WO 97/45106 PCT/US97/09065 -104- a unique band pattern specific to the DNA of subjects suffering from multiple myeloma; f. preparing DNA obtained from a sample of a subject for diagnosis by steps and g. comparing the detected band pattern specific to the DNA obtained from a sample of subjects suffering from multiple myeloma from step (e) and the DNA obtained from a sample of the subject for diagnosis from step to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.
74. The method of claim 72 or 73, wherein the size fractionation in step is effected by a polyacrylamide or agarose gel. The method of claim 72 or 73, wherein the detectable marker is radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.
76. A method of claim 62 which comprises: a. obtaining RNA from the sample of the subject suffering from multiple myeloma; b. separating the RNA sample by size fractionation; c. contacting the resulting RNA species with a nucleic acid probe capable of specifically WO 97/45106 PCT/US97/09065 -105- hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-l protein, wherein the sequence of a nucleic acid molecule encoding a MUM-I protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 and labeled with a detectable marker; d. detecting labeled bands which have hybridized to the RNA species to create a unique band pattern specific to the RNA of subjects suffering from multiple myeloma; f. preparing RNA obtained from a sample of a subject for diagnosis by steps and g. comparing the detected band pattern specific to the RNA obtained from a sample of subjects suffering from multiple myeloma from step (d) and the RNA obtained from a sample of the subject for diagnosis from step to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.
77. A method of claim 63 which comprises: a. obtaining RNA from the sample of the subject suffering from multiple myeloma; b. separating the RNA sample by size fractionation; WO 97/45106 PCTIUS97/09065 -106- c. contacting the resulting RNA species with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-2 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 1 and labeled with a detectable marker; d. detecting labeled bands which have hybridized to the RNA species to create a unique band pattern specific to the RNA of subjects suffering from multiple myeloma; f. preparing RNA obtained from a sample of a subject for diagnosis by steps and g. comparing the detected band pattern specific to the RNA obtained from a sample of subjects suffering from multiple myeloma from step (d) and the RNA obtained from a sample of the subject for diagnosis from step to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.
78. The method of claim 76 or 77, wherein the size fractionation in step is effected by a polyacrylamide or agarose gel.
79. The method of claim 76 or 77, wherein the detectable marker is radioactive isotope, enzyme, dye, biotin, WO 97/45106 PCT/US97/09065 -107- a fluorescent label or a chemiluminescent label. The method of either of claim 72 or 76, wherein multiple myeloma associated with the expression of a specific human MUM-1 is diagnosed.
81. The method of either of claim 73 or 77, wherein multiple myeloma associated with the expression of a specific human MUM-2 is diagnosed.
82. An antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-1 protein so as to prevent overexpression of the mRNA molecule.
83. An antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-2 protein so as to prevent overexpression of the mRNA molecule.
84. An antisense oligonucleotide having a sequence capable of specifically hybridizing to the cDNA molecule of claim 23.
85. An antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA molecule of claim 29.
86. An antisense oligonucleotide having a sequence capable of specifically hybridizing to the RNA molecule of claim
87. An purified MUM protein.
88. A purified MUM protein, wherein the MUM protein is WO 97/45106 PCT/US97/09065 -108-
89.
90.
91.
92.
93.
94.
96.
97. MUM-1 protein. A purified human MUM-1 protein of claim 88. An antibody directed to a purified MUM-1 protein. An antibody capable of specifically recognizing MUM- 1 protein. An antibody of claim 91, wherein the MUM-1 protein is a human MUM-1 protein. A purified MUM protein, wherein the MUM protein is MUM-2 protein. A purified human MUM-2 protein of claim 93. An antibody directed to a purified MUM-2 protein. An antibody capable of specifically recognizing a MUM-2 protein. An antibody of claim 96, wherein the MUM-2 protein is a human MUM-2 protein. An monoclonal antibody of any one of claims 90, 91 and 92. An monoclonal antibody of any one of claims 95, 96, and 97. A pharmaceutical composition comprising an amount of the oligonucleotide of any one of claims 82, 84, and 81 effective to prevent overexpression of a human MUM-1 protein and a pharmaceutically
98.
99.
100. 109 acceptable carrier capable of passing through a cell membrane.
101. A pharmaceutical composition comprising an amount of the oligonucleotide of any one of claims 83, 84, 85 and 81 effective to prevent overexpression of a human MUM-2 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.
102. A method according to claim 1 substantially as hereinbefore described with reference to any one of the examples.
103. An isolated nucleic acid molecule of claim 21 substantially as hereinbefore described with reference to figure o O
104. A method according to claim 62 substantially as hereinbefore described.
105. A method according to claim 63 substantially as hereinbefore described. o 6 00